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+{"text": "PLoS Pathogens, vol 1, issue 4, DOI: 10.1371/journal.ppat.0010039In ts Mutant Phenotype\u201d:In the Results section, under the heading \u201cIdentification of Mutations Responsible for the The reversion of nucleotide 19288 in the Alb17Rs virus results in the substitution of Tyr with Ser, and not with Arg as originally stated.ts Mutants\u201d:Also in the Results section, under the heading \u201cPhenotypes of the MHV-A59 Virus-infected cells were labeled in the presence of 100 \u03bcg per ml of cycloheximide, and not 10 \u03bcg per ml as originally stated."}
+{"text": "Association between NE-FDP and leukocyte content of thrombi is evidenced by a significant Pearson correlation coefficient of 0.71 (p\u00a0=\u00a00.00002). Cluster analysis reveals two classes of thrombi according to NE-FDP, leukocyte and platelet content and also two according to NE-FDP, leukocyte and fibrin content. When NE-FDP, fibrin and platelet content is normalized to the leukocyte count in the same thrombus, clusters with platelet-related thrombolytic resistance (inversely related NE-FDP and platelet content) and advanced cell-dependent thrombolysis (inversely related NE-FDP and fibrin content) are identified. These distinct patterns of thrombus constituents are snapshots of characteristic stages in the cell-dependent thrombolysis, which indicate a clot-stabilizing role for platelets in this process similar to their impact on the plasmin-dependent lysis.Leukocytes invade newly formed thrombi through interactions with platelets and fibrin and later contribute to the removal of fibrin deposits mainly through the action of neutrophil elastase. The present study attempts to express in quantitative terms the impact of neutrophils on the lytic processes in obliterative thrombi based on the local presence of elastase-specific fibrin degradation products (NE-FDP) in relation to the leukocyte, platelet and fibrin content of thrombi. Immunofluorescent detection of fibrin, NE-FDP and platelet antigens was performed in sections of thrombi from 28 patients subjected to thrombectomy in combination with DNA-staining for identification of nucleated cells. The digitalized fluorescent microscopic images were decomposed according to the color channel of each thrombus constituent. The integrated intensity values for all thrombus constituents were statistically evaluated with correlation, hierarchical agglomerative clustering , Hotelling's\u00a0T IIb\u03b23 (GP IIb/IIIa) activation by soluble agonists in vivo contribution of NE to the lytic process in thrombi. The present study was designed to express in quantitative terms the impact of neutrophils on the lytic processes in obliterative vascular thrombi based on the presence of NE-specific FDPs in the thrombus structure in relation to the leukocyte, platelet and fibrin content of thrombi.Thrombus growth at sites of vascular injury or over atherosclerotic plaques is initiated by the shear-dependent platelet aggregation involving the priming adhesive role of the platelet glycoprotein (GP) Ib receptor and subsequent stabilization of the plug through the integrin \u03b1Twenty-eight patients  subjected to thrombectomy were enrolled in the study. Twenty-five of them had obliterative thrombosis localized in large arteries  based on atherosclerosis in 22 cases and related to diabetes mellitus in 6 patients (in 11 cases the thrombus was in a previously implanted graft). Three patients had venous thrombosis . With the exception of the youngest patient with renal vein thrombosis, who had thrombophilia related to systemic lupus erythematosus, no other inherited or acquired thrombophilic state could be diagnosed in the examined group. At the time of thrombectomy all patients received heparin treatment, whereas in their history 12 patients were treated with low-molecular weight heparin, 17 patients with anti-aggregatory drugs and 7 patients with oral anticoagulant. In 2 cases clot lysis with tissue-type plasminogen activator was attempted before the thrombectomy. Written informed consent was obtained from all patients and the study protocol was approved by the institutional and regional ethical board.2 in E3000 Critical Point Drying Apparatus . The specimens were mounted on adhesive carbon discs, sputter coated with gold in SC7620 Sputter Coater  and images were taken with scanning electron microscope EVO40 .Immediately after the surgery, 5\u00a0\u00d7\u00a05\u00a0\u00d7\u00a010\u00a0mm pieces of thrombi were placed into 10\u00a0ml 100\u00a0mM Na-cacodylate pH 7.2 buffer for 24\u00a0h at 4\u00a0\u00b0C. Following repeated washes with the same buffer thrombi were fixed in 1\u00a0v/v% glutaraldehyde for 16\u00a0h. The fixed thrombi were dehydrated in a series of ethanol dilutions (20 \u2013 96\u00a0v/v%), 1:1 mixture of 96\u00a0v/v% ethanol/acetone and pure acetone followed by critical point drying with COAfter surgery, the removed thrombus samples were frozen immediately at \u2212\u00a070\u00a0\u00b0C and stored until examination. Cryosections (6\u00a0\u03bcm thickness) of thrombi were attached to silane-coated slides. Sections were fixed in acetone at 4\u00a0\u00b0C for 10\u00a0min and air-dried for 5\u00a0min at room temperature. After further fixation in 4\u00a0w/v% paraformaldehyde (pH 7.0) at 4\u00a0\u00b0C for 10\u00a0min, the sections were washed in 10\u00a0mM Tris-HCl, pH 7.4, containing 150\u00a0mM NaCl (TBS) for 5\u00a0min, followed by incubation in 100\u00a0mM Na-phosphate 100\u00a0mM NaCl pH 7.5 buffer (PBS) containing 5\u00a0w/v% bovine serum albumin to eliminate nonspecific binding of antibodies. For double immunostaining, sections were first incubated overnight at 4\u00a0\u00b0C in 2\u00a0\u03bcg/mL mouse anti-human NE-digested FDP monoclonal antibody IF-123  Thrombus images acquired at 808\u00a0\u00d7\u00a01104 pixel resolution were saved in JPEG format. All image pixels were allocated to the whole area of the thrombus section so that images did not display any useless information. Image processing was performed by self-devised Matlab scripts running in Matlab environment . RGB images have three channels  according to the three primary colours of light and triple system of notation was applied: the NE-degraded FDPs were allocated to the red channel, the fibrin or platelets were green and nuclei were blue in the same thrombus section. Thus, the separation of the thrombus constituents was based upon the image channels. After importing the thrombus files into the Matlab software environment the full colour RGB images were converted to double-precision images for processing. This process generates three individual monochromatic images representing the red, green, and blue components of the original image. This method enabled automatic and bias-free decomposition of the thrombus image and spatial localization of the thrombus constituents. The constituent intensities as a function of the space coordinates are illustrated on 3D plots in 2 and F-statistics was implemented Statistical calculations were implemented using Statistica 8.0 . Linear regression model was applied for the quantification of the association between the neutrophil cell count and the NE-digested FDP content of the same thrombus sample on one side, and between the neutrophil cell count in the thrombus and in the blood samples of the same patient on the other. In order to identify similarity classes of patients based on the processes going on in the thrombi, we used hierarchical, agglomerative clustering analysis applying Ward's amalgamation rule and Euclidean distances SEM images of thrombi provided evidence of leukocytes forming pseudopodia in-between the fibrin fibers A. LeukocBased on the fluorescent intensity values for NE-digested FDPs and nuclear DAPI staining of sections from surgically removed thrombi , the assa priori hypotheses about the phenomena under investigation, but rather lets the data speak. Thus, this analytic approach has definite advantages under the clinical conditions of our study, when little or no objective data are available about the age of thrombi. When the amount of elastase-digested fibrin, leukocyte content and platelet GPIIb/IIIa antigen were used for classification of thrombi, two main clusters (ELP1 and ELP2) emerged and these were verified by the discriminant function analysis. The size of cluster ELP1 is 18 and the cluster is more homogeneous than cluster ELP2, the size of which is 10 =29.5, p\u00a0<\u00a00.00001). Thrombi isolated from patients of cluster ELP2 had significantly higher elastase-digested fibrin (p\u00a0=\u00a00.0005) content than thrombi of patients belonging to cluster ELP1 (2\u00a0=\u00a00.58), but not in cluster ELP1 (R2\u00a0=\u00a00.25)  C-D. This2\u00a0=\u00a036.4 F=11.2, p\u00a0<\u00a00.00009). The two clusters differed both in content of leukocytes (p\u00a0=\u00a00.009) and elastase-digested fibrin (p\u00a0=\u00a00.00000) according to univariate comparisons  E. The siparisons F, while parisons . Thrombi, potential interrelations of the other examined characteristics  can be evaluated on the basis of a parameter, which is independent of the leukocyte effects. To this end we applied normalization of the fluorescent signals by unit amount of leukocyte. Normalized values for NE-FDP, fibrin and platelet antigen were derived by dividing the integrated NE-FDP, fibrin and platelet signal of each thrombus image by their nuclear-stain signal.Because we found that thrombi showed significantly varying leukocyte contentSuch an evaluation points to the role of additional local factors  superimposed on the cell-dependent thrombolysis. Clustering analysis was performed in two duplets of features: 1) normalized NE-FDP and normalized fibrin; 2) normalized platelet antigen and normalized NE-FDP. According to the first set of characteristics three clusters of patients emerged A: REF1, When normalized platelet antigen and normalized NE-FDP were used as characterizing variables in the cluster analysis, we gained insights into the role of platelets in cell-dependent fibrinolysis. Clustering classified the patients\u2019 thrombi into two groups B: REP1 ain vivo. Using antibody raised against the NE-specific FDPs Despite the early histological evidence for the role of neutrophilic leukocytes in the removal of fibrin in vivo association of the local presence of leukocytes and the cell-dependent degradation of fibrin. Because most of the thrombi evaluated in this study were of atherosclerotic origin, the cell-related thrombolysis may be a significant aspect of the resolution of atherothrombotic clots.Simple visual inspection of the images of triple-stained thrombus sections produced the impression of a massive presence of NE-digested fibrin A and co-a priori distinction criteria were used, all interrelations were established with bias-free statistical evaluation of the quantitative data from the complete set of available samples. When thrombi were classified according to the combination of platelet-, NE-FDP and leukocyte-related signals, two definite classes could be identified  were normalized to the leukocyte signal and cluster analysis was performed on the basis of normalized NE-FDP and fibrin  or normalized NE-FDP and platelet values (yielding two classes: REP1 and REP2) . NormaliIn summary, quantitatively significant cell-dependent fibrinolytic activity in obliterative thrombi correlates with the invasion of leukocytes, the thrombolytic activity of which, in addition, shows dynamic interrelation with the thrombus fibrin and platelet content. Our structural data substantiate the need for further studies to clarify the causal interrelations between the fibrin and platelet content on one hand and the cell-dependent thrombolysis on the other.None to declare."}
+{"text": "Heat Alert and Response Systems (HARS) are currently undergoing testing and implementation in Canada. These programs seek to reduce the adverse health effects of heat waves on human health by issuing weather forecasts and warnings, informing individuals about possible protections from excessive heat, and providing such protections to vulnerable subpopulations and individuals at risk. For these programs to be designed effectively, it is important to know how individuals perceive the heat, what their experience with heat-related illness is, how they protect themselves from excessive heat, and how they acquire information about such protections. In September 2010, we conducted a survey of households in 5 cities in Canada to study these issues. At the time of the survey, these cities had not implemented heat outreach and response systems. The study results indicate that individuals\u2019 recollections of recent heat wave events were generally accurate. About 21% of the sample reported feeling unwell during the most recent heat spell, but these illnesses were generally minor. Only in 25 cases out of 243, these illnesses were confirmed or diagnosed by a health care professional. The rate at which our respondents reported heat-related illnesses was higher among those with cardiovascular and respiratory illnesses, was higher among younger respondents and bore no relationship with the availability of air conditioning at home. Most of the respondents indicated that they would not dismiss themselves as \u201cnot at risk\u201d and that they would cope with excessive heat by staying in air conditioned environments and keeping well hydrated. Despite the absence of heat outreach and education programs in their city, our respondents at least a rough idea of how to take care of themselves. The presence of air conditioning and knowledge of cooling centers is location-specific, which provides opportunities for targeting HARS interventions.  The 2001 and 2007 Intergovernmental Panel on Climate Change (IPPC) reports [Epidemiological investigations based on the statistical analysis of death counts and death certificates have found that mortality increases\u2014sometimes very sharply\u2014above the long-term average during heat waves. Such excess mortality is about an order of magnitude larger than directly observed heat mortality  . This information can be collected through surveys of the population. Previous survey work about this topic has, however, been limited. Sheridan  intervieWhat is reported here is a study in five such cities. In early September 2010, at the end of one of the hottest summers on record , we condv. \u201ctreatment\u201d design.) We added two more metropolitan areas that are similar for climate, geography and population\u2014Regina, SK, and Sarnia, ON\u2014to Winnipeg and Windsor, respectively, but had no plans for public outreach or HARS programs. At the time of this survey, no program had been implemented in any of these five cities, and so the purpose of our research was to gather information about pre-program knowledge of heat-related health risks and ways of coping with the heat.  lie in what Bassil et al.  considerBriefly, we find that individuals\u2019 recollections of recent heat wave events were generally accurate. Our Fredericton, NB, sample was perhaps the group that was the most attuned to registering excessive heat, given the generally cool summers in the Atlantic Provinces of Canada. About 21% of the sample reported feeling unwell during the most recent heat spell, but these illnesses were generally minor. Only in 25 cases out of 243 were these illnesses confirmed or diagnosed by a health care professional. As expected, the rate at which our respondents reported heat-related illnesses  was higher among those with cardiovascular and respiratory illnesses. We were surprised that the younger individuals in our sample were, all else the same, more likely to report heat-related illnesses. We conjecture that this may be the result of spending more time outdoors, confusing air pollution-related symptoms with heat-related illnesses, or misclassification of symptoms. Even more surprising, having air conditioning at home was not related to the likelihood of reporting symptoms.Most of the respondents indicated that would adopt common-sense, and medically appropriate, ways of coping with the heat (such as staying in air conditioned environments and keeping well hydrated), if a heat wave started tomorrow, and were especially proactive with children and elderly persons they are in charge of. In general, our respondents\u2019 awareness of measures to protect themselves from the heat suggest is consistent with the figures reported in  for an eThe remainder of this paper is organized as follows. Physiological adaptation takes place in the presence of excessive heat, and individuals become acclimated in ways that depend on age, gender, health status and cardiovascular fitness levels . In addiAt many locales, adaptation to excessive heat is further enhanced by government programs. Heat/Health Warning Systems (HHWSs) are considered a promising public health tool to reduce the adverse impacts of excessive heat on human health. Briefly, they consist of (i) preparations before the onset of excessive heat; (ii) meteorology-based warning systems; (iii) rapid and coordinated actions during heat waves; (iv) criteria and procedures for deactivating the plan, and (v) evaluations following the response activities and outcomes ,30.In the US, the National Weather Service (NWS) has been issuing excessive heat advisories, watches and warnings since 1993 ,32. The et al. [City heat response program may issue alerts to the population based on the NWS heat advisories, watches and warning, or on alternate criteria more closely linked to the vulnerability of the local population. Systems of this kind are in place in many Midwestern and eastern US cities, and Ebi et al.  estimateetc.) and identifies individuals at risk.In Canada, HHWWs are termed Heat Alert and Response Systems (HARSs). The Heat Advice Program in Canada  providesClean Air Partnership  reviews The various cities and communities within these metro areas adopted a variety of triggers for issuing advisories or warnings. These criteria include (i) spatial synoptic criteria; (ii) a humidex threshold; and (iii) minimum and maximum temperature thresholds. In 2000 the City of Toronto adopted a spatial synoptic classification system developed by Larry Kalkstein and Scott Sheridan at the University of Delaware. This method is location\u2010specific and works by identifying air masses or weather types historically associated with increases in mortality at a given locale. Unlike other watch-warning systems, this method takes into account the negative impact of several consecutive days of oppressive weather, as well as the fact that heat waves earlier in the year are more dangerous than those in late summer. Alerts are issued when oppressive weather is forecast and the likelihood of excess mortality is determined to exceed 65% (Heat Alert) or 90% (Extreme Heat Alert). Because it is based on the likelihood of excess mortality, as opposed to predicted excess mortality counts, the Toronto system is different from similar systems developed by Kalkstein and Sheridan for several US cities. The Peel region in the GTA adopted a spatial synoptic system in 2006. The Kalkstein-Sheridan system has advantages and disadvantages. On the one hand, it caters to local conditions and to the characteristics of the local population. On the other hand, it is a complex system that relies on a large amount of data and is not easily calculated or verified by third parties. There are concerns that alerts and warnings are called too often. There are also challenges with implementing the system in smaller jurisdictions: The smallest metropolitan area for which a synoptic classification system has been developed is approximately 500,000 people. Other communities in the GTA rely on humidex thresholds, a measure that combines heat and humidity to reflect perceived temperature. Alerts are typically issued when the maximum daily humidex value is expected to exceed 40 \u00b0C, or when humidex values are expected to exceed 36 \u00b0C, for an extended period of time (3 days). Potential limitations of humidex-based triggers include that this system does not account for variability in human responses to heat, acclimatization over the summer months or the effects of several consecutive days of high heat and warm nights. Heat thresholds based on the humidex index are not targeted to specific populations but assume that temperature affects all people equally, regardless of the time of year, geographic location or heat wave duration.In Montreal, a heat alert is triggered by a forecast of three consecutive days with lows of 20 \u00b0C or greater and highs of 33 \u00b0C or greater. Thresholds are geographically specific and take into account regional characteristics. In sum, hot weather response plans vary in scope, detail and precision, but typically consist of one or more of the following components: Procedures for alerting municipal staff, community agencies and the public to the occurrence of extreme heat;Procedures to communicate to the public and organizations that work with at-risk groups the health risks associated with extreme heat and heat\u2010safety information; andProcedures for rolling out public health intervention activities that typically include, but are not limited to, opening cooling centers and extending the operating hours of municipal swimming pools and other facilities.The Clear Air Partnership document  emphasizThis latter point underscores that the effect of excessive heat outreach programs and alerts presumably depends on the knowledge that people already have about risks and precautions. Earlier research has also examined and proposed explanations for how people process hazardous weather risk communication and alerts, including decision heuristics that undermine the effectiveness of emergency warnings. A recent investigation in Canada is Silver and Conrad , who stuetc., it is unclear if its findings apply to heat wave situations. Kalkstein and Sheridan [Because much of this research is focused on weather hazards that imply losses to property and \u201caccidental\u201d deaths due to floodwaters, collapsing buildings, fires, Sheridan  conducteSheridan  conducteOur survey questionnaire is comprised of five sections and is reported in the Next, we ask the respondent if he or she experienced any adverse health effects during this heat event, and how severe this heat-related illness was. We further inquire whether it was diagnosed by a health care professional, and whether it interfered with normal daily activities . For policy purposes, it is important to establish whether people heed announcements and the weather forecast in advance of heat wave events, so we ask respondents if they knew ahead of time that it would be very hot, and how they found out. We investigate individual opportunities for coping with the heat by asking respondents what they would do if a three-day heat wave started tomorrow. In three separate questions, we ask people what they would do to take care of themselves, their small children, and elderly family members, relatives, friends or neighbors. Response options include staying inside, using various cooling devices, drinking plenty of water, going to a swimming pool or other body of water, going to the movies or the shopping mall because these places are usually air conditioned, and others. We also inquire if the respondent has heard of cooling centers in his city or elsewhere, and has ever used one, and how he would find out if one or more exist in his city. The questionnaire was self-administered on-line by respondents in five metropolitan areas in Canada see . These cRespondents were recruited from the IPSOS i-say panel, and the sample was to follow quotas for city, gender and age in proportion to the population shares. The survey took place on 2\u201318 September 2010, with the majority of the questionnaires being completed within the first ten days of that month. We received a total of 1141 completed questionnaires. Women accounted for 55% of the sample, and the distribution of the respondents by age was similar across cities. Overall, 18.5% of the respondents were aged 25\u201334, 45% were aged 35\u201354, 16% were aged 55\u201364, 15% were aged 65\u201374, and 5.5% were 75 and older.About one-third of the sample has a college degree or equivalent education, or has done graduate work. About 57% work full- or part-time, 3.77% are looking for work, and students account for less than 4% of the sample. Homemakers and retired persons account for 6% and 28.57% of the sample, respectively. The median household income is between CAN$60,000 and CAN$70,000 (2010 CAN$).Recent epidemiological research emphasizes the importance of good cardiovascular fitness during heat waves. For this reason, we elicited information about the respondent\u2019s health status. The respondents described themselves as being in good (34.88%), very good (37.42%) or even excellent health (14.27%) compared to others the same age. Only 14% of the respondents said that they were in fair or poor health.More details about the respondent\u2019s cardiovascular and respiratory health are displayed in When attention is restricted to the elderly persons that the respondent takes care of, 44.17% of these persons have a chronic cardiovascular or respiratory condition. A total of about 64% of them have some form of mobility impairment . Very few of the respondents  received regular deliveries of meals, groceries, and medicines at home. Six received regular doctor visits at home, and no one reported living in nursing homes with assisted living.Over 95% of the respondents had spent the summer of 2010 in their normal area of residence . Graphs of May\u2013September 2010 daily average and maximum temperatures in each city are displayed in When we asked them whether there were times in the summer of 2010 (defined as 1 June to 15 September 2010) when the weather felt extremely hot, 84.27% of the respondents told us that this was the case. The number of such heat episodes (where an \u201cepisode\u201d was two or more days) ranged from 1 to 100, for an average of 7.45. The median was 4 episodes. The survey questionnaire asked respondents to pinpoint the periods between 1 June and 15 September 2010, when they experienced extremely hot weather, and, as shown in As shown in When asked to describe the most recent heat spell experienced in their area, only 8.53% agreed that it was \u201cvery hot, but dry.\u201d The most frequently selected category was \u201cvery hot and humid\u201d (83.68%), 13.66% chose \u201cthe temperature was not excessively high, but it was extremely humid\u201d, about 18% bemoaned the lack of a breeze, and 23.52% said that \u201cthe night was too warm\u201d. About one-third of the respondents considered this episode \u201cunusual for this time of the year\u201d. The share of respondents who regard this heat spell as unusual varies across location, ranging from 17.11% in the Regina, SK, area to 46.90% in Sarnia, ON, and 70.19% in Fredericton, NB\u2014the normally cool city with an unusually hot early September. How did our respondents cope with this particular heat spell? About 58% spent most of the time in an air-conditioned environment, 31% stayed inside, almost 50% stayed hydrated, 9% spent most of the time at a swimming pool or another body of water, and 11% spent most of the time in the shade. Some respondents had to be outside because of their job (7.6%), and 13.76% said that they did nothing in particular because they enjoy the heat or are not sensitive to it.Did this particular heat episode affect our respondents\u2019 health? Twenty percent of the respondents said it did. We further asked these respondents (and those who were not sure if they had been sick because of the heat) to describe their symptoms. The responses provided by these 243 respondents are summarized in We note that out of the 84 people who reported a \u201cmild heat exhaustion,\u201d only in 4 cases was such a condition diagnosed by a health care professional. Of the 9 reported \u201csevere heat exhaustion\u201d cases, only 3 were confirmed by a health care professional. The ages of these persons ranged from 51 to 68, and the average was almost 58. Of the 14 cases of cardiovascular or cerebrovascular symptoms, 7 were diagnosed by a health care professional. Fourteen people had \u201cother\u201d illnesses or symptoms diagnosed by a health care professional and 44 people went undiagnosed.In sum, 25 people had their illnesses diagnosed by a health care professional. Eleven (44%) regarded these illnesses as minor, in the sense that they really did not change their daily routine. Nine (36%) considered them significant, because they had to make changes to daily routine, and for five people (20%) they were major, because they had to go to the hospital, health clinic or similar facility. We checked whether the incidence of illness during the most recent heat spell varied across the study areas, and indeed it did. As shown in We had expected the illness rate during the latest heat episode to be highest among the elderly, but this expectation is not borne out in the data. Epidemiologic studies have linked heat-related illnesses to poor cardiovascular and respiratory health status, and so we are not surprised to see that, as shown in Out of the 188 people who said that they experienced this illness in 2010 (47.72% of persons who ever experienced a heat-related illness), 162 indicated that they got sick in July or August. A total of 6 people said that they experienced this illness in January through May 2010\u2014presumably during the early heat wave at the end of May\u2014and 12 people in September.Air conditioners and other cooling devices have a strong protective effect during heat waves. This section of the paper reports about the rate of penetration of home air conditioning and examines the association between air conditioning and heat-related illness. Of the other cooling devices, fans are common (87% of the sample), but only 6 respondents have a swamp cooler. Out of the 210 respondents who live in housing other than single-family homes or semi-attached homes, 78 said that there is a lobby or common area with A/C, 118 did not have such common area with A/C, and 14 did not know. Our survey data suggest that when the most recent heat spells occurred, most people (87.38%) were aware that it was going to be extremely hot ahead of time. They had heard it on the weather forecast  , or fromWe asked respondents what they would do if they heard that, starting the next day, there would be a three-day heat wave. As shown in Taking the children to a pool or a body of water was a popular option for parents (53%), but was selected much less frequently for oneself (14%) or for one\u2019s elderly charges (2.9%). Movies and shopping (both air-conditioned places) were not popular selections for the respondents or the elderly, but 13% of the parents indicated that they might take (or send) their children to a shopping mall. The option of going out of town was selected by relatively few respondents. In general, respondents tended to be somewhat more proactive with their children and any elderly persons they take care of than for themselves.The respondents seemed realistic in their assessment of what they would do to protect themselves from excessive heat. As shown in Regarding \u201ccooling centers,\u201d 14.20% of our respondents had heard of them in their city, 29.27% had heard of cooling centers in other cities, and almost 59% had never heard of them at all. Only 12 respondents (or 1% of the sample) said that they had used a cooling center before. Three of these respondents live in Regina, 3 in Winnipeg, 1 in Sarnia, 3 in Windsor and 2 in Fredericton.In Winnipeg, 68% of the sample had never heard of cooling centers. In Regina, the share of the sample who does not know cooling centers is 71%. The questionnaire also inquired about the respondents\u2019 perceptions about individuals and populations at risk during heat waves. We have conducted a survey of households in five cities in Canada to assess their perceptions of excessive summer heat, experience with heat-related illnesses, and coping mechanisms. The survey was conducted in the summer of 2010. At the time of the survey the cities had not yet implemented formal heat illness outreach program or heat response programs. more likely to report heat-related illness, and air conditioning bore no relationship with the likelihood of experiencing a heat-related illness, whether or not we controlled for the area of residence of the respondent, gender, and existing chronic illness conditions. Since the protective role of air conditioning is well-established, we suspect that result may be due to confusion about the true nature and cause of the illness (heat waves are often accompanied by severe air pollution episodes), misclassification of symptoms, or simply spending time outdoors. Indeed, our probit regressions suggest that, even controlling for respondent characteristics and air conditioning at home, illness rates are somewhat higher at locales where, for any given temperature, the heat index and/or humidex would be higher .Our respondents were generally well aware of the heat spells in that summer, and 21% of them reported having been unwell because of the heat. The majority of these episodes of heat-related illness, however, were relatively minor. Our survey confirms the medical and epidemiological literature, in that the respondents who report the highest rates of heat-related illness during the most recent heat wave are those in compromised cardiovascular and respiratory health. Surprisingly, younger respondents were etc.\u2014even though a formal outreach program had not been initiated yet. Most likely the media and other sources have provided plentiful information about heat mitigation. Our respondents seemed especially proactive in seeking such protections for their young children and any elderly they are responsible for. In general, they tended to think that virtually everyone is at risk during heat waves, not just persons in compromised health.When asked how they would protect themselves, their young children and any elderly person that they are in charge of from a hypothetical heat wave that starts tomorrow, our respondents appear to be well aware of common-sense, medically recommended protections, such as staying in climate-controlled environments, keeping hydrated, Our survey also demonstrated that air conditioning is not ubiquitous in Canada: in the Fredericton area, about 47% of our respondents did not have air conditioning in their home, a figure that is in sharp contrast with those in our Ontario cities and Winnipeg. Our respondents were also relatively unaware of \u201ccooling centres,\u201d since 59% had never heard of them and only 1% had used them before. These results and the other findings of this study suggest that outreach initiatives that inform individuals about cooling centers and similar options, and emphasize the importance of limiting exposure to hot and humid conditions, will be especially useful in reducing heat-related illness."}
+{"text": "The pinewood nematode genome encodes at least three distinct acetylcholinesterases (AChEs). To understand physiological roles of the three pinewood nematode AChEs , BxACE-3 in particular, their tissue distribution and inhibition profiles were investigated. Immunohistochemistry revealed that BxACE-1 and BxACE-2 were distributed in neuronal tissues. In contrast, BxACE-3 was detected from some specific tissues and extracted without the aid of detergent, suggesting its soluble nature unlike BxACE-1 and BxACE-2. When present together, BxAChE3 significantly reduced the inhibition of BxACE-1 and BxACE-2 by cholinesterase inhibitors. Knockdown of BxACE-3 by RNA interference significantly increased the toxicity of three nematicidal compounds, supporting the protective role of BxACE-3 against chemicals. In summary, BxACE-3 appears to have a non-neuronal function of chemical defense whereas both BxACE-1 and BxACE-2 have classical neuronal function of synaptic transmission. Acetylcholinesterase  plays a critical role in terminating nerve impulses by hydrolyzing the neurotransmitter, acetylcholine (ACh) in the cholinergic nervous system of most animals Caenorhabditis elegans, a free-living nematode, has four ace genes encoding different AChE types . Each AChE showed different pharmacological properties Heterodera glycines, Meloidogyne arenaria and M. incognitaBursaphelenchus xylophilus, a serious phytopathogen to pine trees in several countries in vitro. BxACE-1 and BxACE-2 showed similar biochemical properties, including the catalytic efficiency and the substrate and inhibitor specificities, to those of other nematodes. Unlike BxACE-1 and BxACE-2, which appear to play common but non-overlapping synaptic functions, BxACE-3 exhibited several unique features. First, no GPI-anchoring sequence or the H-type hydrophobic sequence was predicted from cDNA, suggesting the soluble nature BxACE-3. Second, BxACE-3 showed the highest transcription level but lowest catalytic efficiency, indicating that it may not function in postsynaptic transmission. Third, the sensitivity to various anti-cholinesterase inhibitors was much lower in BxACE-3, showing unusual inhibition properties compared to typical neuronal AChEs. Based on these findings, it was hypothesized that BxACE-3 is not likely involved in postsynaptic transmission but rather has non-neuronal functions In our previous study, three AChEs  were identified from the pinewood nematode, in vitro inhibition profiles in the presence or absence of BxACE-3 and the organophosphate inhibition sensitivity of the nematodes when expression of BxACE-3 was knocked down by RNA interference (RNAi). We provided some lines of evidence that BxACE-3 has a role as bioscavenger against anti-AChEs, thereby providing non-neuronal functions of chemical defense. In addition, we demonstrated that BxACE-3 interacts with pine resin terpenes and postulated that B. xylophilus has evolved the chemical defense system via BxACE-3 against the endogenous anti-AChE terpene compounds. In this study, to further understand the physiological roles of BxACEs, BxACE-3 in particular, we investigated their tissue distribution patterns via immuohistochemistry, B. xylophilus was collected from the Jinju in Korea by the method described in previous study Botrytis cinerea cultured on PDA plates  at 28\u00b0C for up to a week. Fresh nematode washed by M9 buffer Escherichia coli BL21(DE3). Immunogens were expressed by IPTG induction, and then purified using a His-tag column. The purified antigens were injected into a rabbit three times, and BxACEPabs were obtained . BxACEPabs were purified by an affinity chromatography column using the respective antigens.Recombinant BxACEs were expressed by baculovirus system described in previous studies and their activity was verified by kinetics B. xylophilus was used for immunohistochemistry of BxACEs. A whole-body immunohistochemistry procedure was conducted using the tube-fixation protocol according to Wormbook B. xylophilus was based on other model nematodes, including C. elegans and Ascaris suum, because there is a remarkable conservation of neuronal morphology in nematodes despite large differences in size and habitat MGPS of B. xylophilus were extracted using an ultrasonicator, Sonifier 450  in 0.1 M Tris-HCl (pH 7.8) containing 20 mM NaCl in the presence or absence of 0.5% Triton-X. Native PAGE was conducted at 120 V for 90 min at 4\u00b0C, and the gel was electro-blotted to a nitrocellulose membrane . Each BxACE was detected using BxACEPabs.To determine whether BxACEs were associated with the membrane, proteins from Acetylthiocholine iodide (ASChI), 5,5\u2032-dithiobis(2-nitro-benzonic acid) (DTNB), bovine serum albumin (BSA), \u03b1-pinene and limonene were purchased from Sigma-Aldrich. Insecticides, including dichlorvos, chlorpyrifos-oxon, and carbofuran, were purchased from ChemService .in vivo state of BxACEs, and used in an inhibition assay. Five concentrations of dichlorvos, chlorpyrifos-oxon and carbofuran were directly added to the mixture of BxACE-1 and BxACE-2. Otherwise, the inhibitors were first pre-incubated with BxACE-3 for 10 min and then added to the mixture of BxACE-1 and BxACE-2. In the final BxACE mixture, the molar ratio of BxACE-1:BxACE-2:BxACE-3 was 6:1:18, on the basis of their respective transcript levels in the PGPS 50 was calculated using the Polo Plus program . An equality analysis between different treatments was conducted using GLM repeated measures ANOVA in the SPSS program .To investigate the effects of BxACE-3 on the inhibition of BxACE-1 and BxACE-2, BxACE-1 and BxACE-2 were mixed in a ratio of 6:1, based on their transcription levels in the pine-grown propagative stage (PGPS) To determine the anti-AChE activity of \u03b1-pinene and limonene, each BxACE sample was incubated with six serially diluted concentrations of the test chemical in 0.1 M Tris-HCl buffer  containing ASChI  and DTNB . The remaining AChE activity was measured as described above.TM RNAi designer . The pQE30 plasmid sequence, which does not exist in B. xylophilus, was used as the negative control. A PCR product was amplified using Ex Taq polymerase , from which double-strand RNA (dsRNA) was prepared using MEGAscript RNAi Kit . Approximately 300\u2013500 mixed-stage nematodes were soaked in 20 \u00b5l RNAi solution (0.25x Mg2+-free M9 buffer) containing 3 mM spermidine (Sigma-Aldrich), 0.05% gelatin (Sigma-Aldrich), lipofectin (Invitrogen) and dsRNA (4 \u00b5g/\u03bcl).For the amplification of DNA templates used for dsRNA synthesis, primers containing T7 polymerase promoter sequence  were des50 value, unpublished) by soaking at RT and mortality was determined at 24 hr post-treatment. Total RNA was extracted from remaining nematodes using TRI reagent  and used for qPCR to determine the extent of target gene knockdown. The qPCR was conducted by the procedure described in previous studies Chlorpyrifos-methyl and fenamiphos were purchased from ChemService. After incubation in dsRNA solution for 1 day at 30\u00b0C, approximately 50\u2013100 nematodes were treated with a diagnostic dose of chlorpyrifos-methyl, dichlorvos and fenamiphos  in the presence of BxACE-3 as determined by GLM repeated measures ANOVA (p<0.05)  . In partC. elegans in which the transcript level of orthologous AChE was similar to other AChEs as estimated by Northern blotting BxACE-3 exhibited the highest transcription level in all of the life stages 50 against nerve agents, demonstrating its protective function In this sense, BxACE-3 resembles the vertebrate butyrylcholinesterase , which serves as an effective sequestering agent against toxins or toxicants before they arrive at their target sites Bxace-3 dsRNA (\u2013Bxace-3) was 48% suppressed compared to the nematodes treated with pQE30 dsRNA (pQE30\u2013) (\u2013Bxace-3 nematodes (data not shown). In bioassays using chlorpyrifos-methyl, dichlorvos and fenamiphos, however, the mortality of \u2013Bxace-3 nematodes  was 1.3\u20132.0-fold higher than that of pQE30\u2013 nematodes , demonstrating that knowndown of Bxace-3 increased the mortality of B. xylophilus (BxACE-3 transcription in the nematodes treated with pQE30\u2013) . Neverthlophilus . These lB. xylophilus to the toxic secondary compounds of pine tree hosts may have driven BxACE-3 to adapt to such a chemical defense function. B. xylophilus spends most of its life inside pine tree except for migration via insect vectors and must counteract against various physical and chemical defenses by the host pine tree, particularly in the propagative stage. Pine resin is a major defense compound against various pests, including insects, nematodes and fungi B. xylophilus has evolved the chemical defense system based on BxACE-3 against the anti-AChE terpene compounds.Constant exposure of the monophagous C. elegans ACE-3 shows different expression patterns from BxACE-3 B. xylophilus.To test this hypothesis, AChE inhibition assays using \u03b1-pinene and limonene were conducted. \u03b1-pinene inhibited BxACE-1 in a dose-dependent manner . BxACE-1B. xylophilus, both BxACE-1 and BxACE-2, which have relatively high catalytic activities, are involved in the postsynaptic transmission, whereas BxACE-3 appears to adapt to a non-neuronal role of chemical defense against xenobiotics. The role of BxACE-3 as a bioscavenger provides important insights into the functional diversification of AChE.AChE is presumed to be generated before cnidarians, but ancient AChE appears to have non-neuronal functions because AChE of cnidarians shows a low catalytic activity and is mostly expressed in non-nervous tissues Figure S1The cross activity assay of three BxACEPabs against recombinant BxACEs expressed by baculovirus system through Western Blot. Each BxACEPab shows no cross activity against other BxACEs.(PDF)Click here for additional data file.Table S1The list of primers for synthesis of dsRNA used in RNAi.(PDF)Click here for additional data file.Table S250 of several BxACE mixtures against three nematicidal reagents.The IC(PDF)Click here for additional data file."}
+{"text": "The International Society for Computational Biology (ISCB) honors the achievements of an early- to mid-career scientist with the Overton Prize each year. The Overton Prize was instituted to honor the untimely loss of Dr. G. Christian Overton, a respected computational biologist and founding ISCB Board member. Winners of the Overton Prize are independent investigators in the early to middle phases of their careers who are selected because of their significant contributions to computational biology through research, teaching, and service.ISCB is pleased to recognize Dr. Curtis Huttenhower, associate professor of computational biology and bioinformatics at the Harvard T. H. Chan School of Public Health, as the 2015 winner of the Overton Prize. Huttenhower will be presenting a keynote presentation at the 2015 Annual International Conference on Intelligent Systems for Molecular Biology/European Conference on Computational Biology (ISMB/ECCB) in Dublin, Ireland, in July 2015.Curtis Huttenhower  has viviHuttenhower graduated in 2000 from the Rose-Hulman Institute of Technology, where he majored in computer science, chemistry, and math. He remembers being drawn to the physical sciences as well as computation, but he also admitted that despite his interest in the natural sciences, he was at first discouraged from studying these fields because he was dreadful at memorization. He recounted, \u201cI was, and remain, absolutely terrible at memorizing lists of facts. That kept me out of biology for a long time. It's a rare student who enjoys the toughest parts of general biology or chemistry, memorizing gene and chemical names. Organic chemistry was all about rules rather than facts, though\u2014generalizable principles. That got me into biochemistry, and the combination of problem solving in the wet lab and problem solving in the dry lab, by way of bioinformatics, was a lot of fun.\u201dAlthough Huttenhower graduated with excellent undergraduate academic performance, he was initially rejected from all the graduate programs he applied to, and he found himself going in a very different direction by taking a software development job with Microsoft. In retrospect, though, he considers his two years at Microsoft as an invaluable experience, during which he learned the importance of management and testing infrastructure and the value of standard operating procedures and standardized methods for organizing computational projects. Huttenhower also sees value in this experience as he advises trainees about career options in academia and industry. \"Particularly given the challenges of the modern life science careers,\" he mentions, \"it's important to remember how productive and enjoyable industry work can be, and that neither of the two options are intrinsically better or worse than the other.\"Huttenhower was finally accepted into a graduate program in computational linguistics at Carnegie Mellon University after two years of applications, and he has a new perspective on this process now. \u201cServing on admissions committees a decade later,\u201d he said, \u201cI understand better now what a challenging process it is.\u201d Dannie Durand was Huttenhower\u2019s research advisor, and he credits her for his current career path. He recalled, \u201cShe was a joy to work with and tremendously enthusiastic about bioinformatics, and the enthusiasm was catching. By the time I finished my MS, I was applying for PhD programs in computational biology rather than language processing. She and Russell Schwartz were, and still are, fantastic motivators of new students entering the field.\u201dHuttenhower then went to Princeton University to pursue his PhD and postdoctoral studies in computer science under the guidance of Olga Troyanskaya. He credited Troyanskaya\u2019s mentorship as elemental to his success, and he described, \u201c[She] created, and has since grown, an exciting and productive lab. In addition to key pieces of basic knowledge in bioinformatics and strategies for a research career, I learned from her how to figure out which scientific problems matter, at least inasmuch as any of us know.\u201dIn 2009, Huttenhower accepted a position as an assistant professor of computational biology and bioinformatics in the Department of Biostatistics at the Harvard T. H. Chan School of Public Health. He considers himself fortunate to have great mentors, especially Owen White, John Quackenbush, and Ramnik Xavier, as he has launched his academic career. Huttenhower has also considered his friendships and collaborations with other junior faculty as crucial to early survival. He sees now that this mutual support and guidance has been pivotal in the success of early career computational biologists. \u201cChad Myers, Matt Hibbs, Florian Markowetz, and Edo Airoldi formed the core of Olga's lab along with me in her first few years as faculty,\u201d he recalled, \u201cand they've all gone on to extremely successful research careers.\"Huttenhower has established a robust research program in his lab, with a large part of his group working on projects related to the National Institutes of Health (NIH) Human Microbiome Project. He considers microbiome research to be an area of particular interest because relatively little is known about microbial communities, the field has a broad and solid foundation in classical microbiology on which to build, and findings in this area may significantly influence human health. He is enthusiastic about translating microbiome research to clinical applications. He said, \u201cThe microbiome represents an untapped new source of possible disease biomarkers and therapeutic interventions. In inflammatory bowel disease and type 1 diabetes, we're trying to determine which changes in the microbiome predict disease onset or inflammatory activity. Even in the worst case, knowing when a flare or seroconversion was becoming likely would allow stronger treatments to be introduced to prevent exacerbation or stave off disease activity. In the best case, the microbiome is modifiable, unlike human genetics. It's difficult to modify, since like cancer it represents a complex system that's evolved to be resistant to change, but the potential is there.\u201dSaccharomyces cerevisiae has been a great model microbial isolate for decades, and we're still learning how to make better wine, beer, and bread, so research into the complexities of mixed microbial communities is unlikely to run out of challenges any time soon!\u201dHuttenhower is excited about his work, and he shares a good problem to have with other life sciences researchers. \u201cLike many scientists in public health,\u201d he said, \u201cwe spend our time working both on translational applications and on interesting basic biology, and at this point in the field as a whole there are many findings from both areas that represent ongoing work with a lot of potential.\u201d The tools developed for his lab\u2019s human microbiome research can also be applied to research on other microbial communities, and there are many open questions in their uses for areas as far-flung as agricultural microbial communities, bioremediation, and the basic biology of microbial interactions. Huttenhower noted, \u201c"}
+{"text": "The aim of this study was to determine the relationship between polymorphisms of peroxisome proliferator activated receptor - PPAR gamma-2  and beta 3-adrenergic receptor - ADRB3 (Trp64Arg) and dietary habits in a group of postmenopausal women who were not under hypolipidemic treatment.Genetic, nutritional and anthropometric parameters were measured in 213 dyslipidemic (LDL \u2265115 mg/dL) and 58 normolipidemic (LDL<115) postmenopausal women. The PCR-RFLP method were used to determine the distributions of selected alleles and genotype frequencies. Dietary intake of basic components and fatty acids was obtained from a 7-day weighed food record and the bio-impedance method was used to determine nutritional status.Nearly 79% of analyzed women were in the firsttime-diagnosed dyslipidemic state. The dyslipidemic subjects were characterized with higher intake of energy, fat, and saturated fatty acids (SFA). The analysis of the same polymorphisms showed association at the P value <0.05 with nutrients  and elevated LDL level. Higher PUFA intake in a group of women with the protective Ala12/X polymorphism did not increase the risk of dyslipidemia even though they were characterized by visceral distribution of fat. The Arg64/X polymorphism and higher intake of energy, fat, and arachidic acid intake (C20:0) were associated with dyslipidemic state.Both nutritional and genetic factors are related to lipid profile. The identification of gene-diet associations is likely to provide useful information about the etiology of postmenopausal dyslipidemia and help in effective treatment. Many European populations, including Poland, represent countries of high risk of cardiovascular diseases (CVD) . Recent Some studies have reported on the combined role of genetic  and lifestyle (such as dietary fatty acid ratio) factors in metabolic disorders  , 11. HowIn this study, 1,423 women, aged 49 to 75 years, were recruited and they underwent standard health checkups at a metabolic outpatient clinic. From this group, we selected postmenopausal women who did not undergo any hypolipidemic or hypoglycemic treatment. After the gynecological interview , the hormonal assessment based on the measurement of follicle-stimulating hormone (FSH) confirmed the postmenopausal period. Women earlier diagnosed with or treated for dyslipidemia, severe cardiovascular diseases, endocrinological disorders, renal or liver dysfunction, cancer or the ones supplemented with minerals/vitamins/fitosterols were excluded from the study. Finally, we selected 271 postmenopausal women, who undergo biochemical, nutritional, and genetic evaluation. The selection was random because PPAR\u03b32 and ADR\u03b23 polymorphisms were unknown at the time of recruitment. The ethical approval was obtained from the respective local Bioethical Commission of Poznan Medical University, Poland, nr 792/09, and the guidelines proposed by the Declaration of Helsinki were followed. Written informed consent was obtained from all subjects before the commencement of the study. Anthropometric measurements were directly taken in accordance with the International Standards for Anthropometric Assessment  by trainAfter 12 hour fasting, venous blood samples were collected from all patients at 7 A.M. Serum samples were taken from clotted  and centrifuged blood . Serum was separated and directly used for the assay. The obtained samples were used for the measurements of FSH levels and plasma lipid profile. The concentration of FSH was measured to confirm the postmenopausal age by chemiluminescence assays (Roche Diagnostics). The lipid profile was assessed with enzymatic colorimetric assays . Low density lipoprotein (LDL) cholesterol was calculated using Friedewald\u2019s formula .According to the recommendation of European Society of Cardiology (ESC) and the European Atherosclerosis Society (EAS) the characteristic SCORE (Systematic Coronary Risk Estimation) was calculated for whole group of women n=271). Estimated SCORE (based on data for no-smoking women aged 60) was within the range of 1%\u20135%. The assessed coronary risk was moderate and thus LDL-cholesterol of 3 mmol/L (less than 115 mg/dL) should be considered as a target value in the analyzed group 71. Estim. ConsideThe food intake was assessed at a 24 hour interval for 7 days and all women were on normal diet . The estimated daily food rations (DFR) enabled the analysis of energy intake, basic nutritional components consumption  and selected fatty acids intake . The results of the questionnaire were analyzed with both the quantitative and qualitative analyzes of the subjects\u2019 daily diets using computer databases for Microsoft Access 2000. The food intake recommendations of the National Institute of Food and Nutrition in Warsaw, Poland, were taken in to account to determine whether the Recommended Dietary Allowances (RDAs) specific for age, sex, ideal body mass, height, and physical activity were fulfilled . DietaryGenomic DNA was isolated from venous blood samples according to the manufacturer\u2019s protocol . Genotypes of the Pro12Ala (rs1801282) and Trp64Arg polymorphism (rs4994) were determined by applying a TaqMan genotyping assay . As a quality-control measure, negative controls and approximately 5% of samples were genotyped in duplicate to check genotyping accuracy. The controls for each of the genotypes of both single nucleotide polymorphisms were run in parallel. An allelic discrimination assay was performed on an ABI7900HT or on CFX96 Touch Real-Time PCR Detection System . C1431T (rs3856806) genotyping was performed using PCR-restriction fragment length polymorphism (PCRRFLP) analysis. Eco72I cleaves the PCR product from wildtype DNA to generate fragments of 127bp and 43bp, but does not cut products containing the variant allele. PCR-digests were analyzed on 2.5% agarose gels. The determination of the ADR\u03b23 variant was performed using the PCR method as described by Sivenius and colleagues . The genThe genotype data were used to construct the haplotypes between the two polymorphisms by using Haploview 4.2 software  to evaluate linkage disequilibrium (LD). Linkage disequilibrium between the single nucleotide polymorphisms used in haplotype analysis was measured using a pairwise D\u2019 statistic. The structure of the LD block was examined with the method proposed by Gabriel and colleagues  using thThe Shapiro-Wilk test was used to determine if the continuous variables were normally distributed. Since the results were not consistent with normal distribution, nonparametric methods were used for the statistical analysis. Continuous data were shown as mean \u00b1 standard deviation. The hypothesis that the differences between analyzed anthropometric and metabolic factors in the analyzed groups were significant was tested by Mann-Whitney U-test. As the number of Ala12Ala homozygotes were small (one lean woman and nine obese women), compared to Pro12Pro homozygotes, they were collapsed with Pro12Ala heterozygotes for all the analyzes. Similarly, C1431T heterozygotes and T1431T homozygotes were collapsed together, as well as Trp54Arg heterozygotes and Arg64Arg homozygotes. Allele frequencies were estimated using the gene-counting method, and an exact test was performed to identify departures from Hardy-Weinberg proportions . Genotype and allele frequencies according to normolipidemic (LDL <130 mg/ dL) and dyslipidemic state (LDL \u2265130 mg/dL) were tested by Mann-Whitney U test. The association of analyzed genotypes with anthropometric and nutritional parameters in normo- and dyslipidemic women was also tested by Mann-Whitney U test. The odds ratio (OR) with a 95% confidence interval (CI) for gene polymorphisms according to normolipidemic (LDL <115 mg/dL) and hyperlipidemic state were determined. A P value <0.05 was regarded as statistically significant. The statistical analyzes were performed with STATISTICA 12  and SPSS 22 .The anthropometric parameters of dyslipidemic and normolipidemic subjects were comparable in both groups . All womSeveral factors were proposed to be associated with dyslipidemia including dietary habits, physical activity, and genetic background , 29-32. The analyzed groups of women  were oveThe intake of energy, as fat and saturated fatty acids were higher in dyslipidemic women, while PUFA/SFA ratio was lower . Mainly In this study, we did not confirm the differences between gene distribution and the presence of dyslipidemia . HoweverDyslipidemic women with Arg64/X polymorphism of the ADR\u03b23 were characterized with higher intake of energy, total fat, and arachidic acid (C20:0). Thus, this polymorphism seems to be related to dyslipidemic state and can increase the lipid disorders if it is associated by higher intake of proatherogenic nutrients. This evidence is confirmed by the study of Asian  and CaucTo our knowledge this is the first study, which showed a genetic associations of ADR\u03b23 and PPAR\u03b32 variants and eating behavior in postmenopausal women with dyslipidemia who did not undergo hypolipidemic treatment. The dyslipidemia was first time diagnosed in nearly 80% of postmenopausal women, which suggest the need for early diagnosis, dietary modification, and/or hypolipidemic treatment in postmenopausal women to prevent CVD development. According to the recommendations of ESC and the European Atherosclerosis Society (EAS) women with LDL \u2265115 mg/ dL should undertake lifestyle interventions including dietary modification and increased physical activity. Therefore, lower intake of fat and SFA and higher intake of PUFA should be recommended. Alongside, the presented results suggest that some polymorphisms together with the selected nutrients are related to dyslipidemia. Therefore, Ala12 (protective) and Arg64 allele (predisposing to dyslipidemia) may play an important role in the treatment strategies in long-term weight changes in viscerally overweight postmenopausal women with newly diagnosed dyslipidemia.Ethics declaration: All experimental procedures were conducted in accordance with the guidelines in the Declaration of Helsinki and approved by the Bioethics Committee of Poznan University of Medical Sciences in Poland.Acknowledgments: B.G.G designed the study and supervised the practical carrying out of the clinical trial; M.M. conduct genetic measurement; E.K. and B.G.G. do statistical analysis; J.P. and J.N. analyzed the data; B.G.G. wrote the manuscript and had the primary responsibility for final content. All authors read and approved the final manuscript.Funding source: This study was supported by the Polish National Science Center (NSC) under grant No. N404 504 638.Conflict of Interest Disclosures: The authors do not have any conflicts of interest"}
+{"text": "Drosophila melanogaster for 13 generations. At the end of the selection procedure, night sleep duration diverged by 9.97 hours in the long and short sleeper populations, and 24-hour sleep was reduced to 3.3 hours in the short sleepers. Neither long nor short sleeper lifespan differed appreciably from controls, suggesting little physiological consequences to being an extreme long or short sleeper. Whole genome sequence data from seven generations of selection revealed several hundred thousand changes in allele frequencies at polymorphic loci across the genome. Combining the data from long and short sleeper populations across generations in a logistic regression implicated 126 polymorphisms in 80 candidate genes, and we confirmed three of these genes and a larger genomic region with mutant and chromosomal deficiency tests, respectively. Many of these genes could be connected in a single network based on previously known physical and genetic interactions. Candidate genes have known roles in several classic, highly conserved developmental and signaling pathways\u2014EGFR, Wnt, Hippo, and MAPK. The involvement of highly pleiotropic pathway genes suggests that sleep duration in natural populations can be influenced by a wide variety of biological processes, which may be why the purpose of sleep has been so elusive.Why do some individuals need more sleep than others? Forward mutagenesis screens in flies using engineered mutations have established a clear genetic component to sleep duration, revealing mutants that convey very long or short sleep. Whether such extreme long or short sleep could exist in natural populations was unknown. We applied artificial selection for high and low night sleep duration to an outbred population of  Drosophila melanogaster to an experimental breeding program. At the end of the breeding program, long sleepers averaged 9.97 hours more nightly sleep than short sleepers. We analyzed whole-genome sequences from seven generations of the experimental breeding to identify allele frequencies that diverged between long and short sleepers, and verified genes and genomic regions with mutation and deficiency testing. These alleles map to classic developmental and signaling pathways, implicating many diverse processes that potentially affect sleep duration.One of the biggest mysteries in biology is the need to sleep. Sleep duration has an underlying genetic basis, suggesting that very long and short sleep times could be bred for experimentally. How far can sleep duration be driven up or down? Here we achieved extremely long and short night sleep duration by subjecting a wild-derived population of  Sleep remains a classic enigma in biology. Intense study has only begun to reveal the physiological needs that sleep might satisfy , 2. One Another possibility is that sleep is crucial for proper development. Babies and young infants spend far more time asleep than their adult counterparts , and intSleep-like behaviors are widely conserved among species , 13\u201315, D. melanogaster as a model for mammalian sleep [D. melanogaster, which lacks the genomic redundancy seen in mammals. Indeed, extremely low sleep duration has been observed in flies with single mutations in Shaker (Sh), sleepless (sss), insomniac (inc), and nicotinic Acetylcholine Receptor \u03b14 (nAChR\u03b14) [Previous work has demonstrated the value and utility of an sleep , 43. Thean sleep \u201351, genoan sleep , quantitan sleep , and genan sleep \u201355 possinAChR\u03b14) , 50, 51.The short generation time of flies makes it possible to combine artificial selection or laboratory evolution with whole-genome sequencing to understand the genetic basis of complex traits. Such a strategy has been used to explore adaptation to different environments \u201360, maleP = 0.0002; P = 0.0344; CVE) because artificial selection, which uses only a subset of each population as parents for the next generation, tends to reduce phenotypic variance [CVE had a significant correlated response to selection for night sleep (P <0.0001), increasing in flies selected for short night sleep and decreasing in flies selected for long night sleep. The estimated realized heritabilities h2, which indicate the degree to which the animals responded to the selection procedure, were relatively high for long-sleepers [h2 = 0.310 \u00b1 0.022 and h2 = 0.238 \u00b1 0.032  for replicates 1 and 2, respectively  (P = 0.7368) and -0.271 \u00b1 0.206 (P = 0.2161) for replicates 1 and 2  constructed from the most extreme long- and short-sleeping lines of the Drosophila Genetic Reference Panel (DGRP). Sleep characteristics were uniform in the outbred population . We selevariance . Interessleepers , 68\u201370; ectively . For sho<0.0001) . In addi 1 and 2 }, suggesP = 0.0248), night average bout length (P = 0.0358), day bout number (P = 0.0121), and sleep latency (P = 0.0005) responded to selection for long or short night sleep, even though we did not select for these changes (CVE (P < 0.0001), night average bout length CVE (P < 0.0001), day bout number CVE (P <0.0001), and sleep latency CVE (P = 0.0401) had a significant correlated response to selection for night sleep , 9 (P = 0.0352) and 10 (P = 0.0455). Short-sleeping females were the most vulnerable, though flies of all populations were less likely to survive the sleep monitoring. Thus, any physiological consequences of being an extreme long or short sleeper did not manifest themselves in either lifespan or egg-to-adult viability, but the reduced survival of short sleepers during the later generations of selection suggests that they might be more susceptible to stress.Sleep is crucial for life, yet its relationship to important life history and fitness traits is not well understood. Several previous mutagenesis screens have noted reduced lifespan in mutants with short sleep duration , 77, 78,PTreatment and PTreatment\u00d7Day < 0.0001 for night sleep in all populations)  . Long-sllations) , their slations) . Short slations) . They dilations) . The reslations) , with a lations) . Short slations) . Their slations) . Intereslations) . Thus, ade novo polymorphisms that might be present in our selected populations; LoFreq detected an additional 258,268 potential polymorphisms . We defined major and minor alleles in our population by summing the allele counts for all polymorphisms across populations for generation zero, the generation prior to the start of selection for night sleep. Minor allele frequency distributions were fairly flat across all five chromosome arms  (We used the Cochran-Mantel-Haenszel (CMH) test to detect significant changes in allele frequency between any two generations for each replicate population ; forms o \u00d7 10\u22128) . We ther \u00d7 10\u22128) . Interes \u00d7 10\u22128) ; 111 of  \u00d7 10\u22128) . Combinide facto LD with the remainder of the population [r2 and minor allele frequency at variant A are specified [r2 \u2265 0.8); this is 1.65% of the possible 161,991,000 pairwise combinations, indicating that LD among the ten DGRP lines is quite low. Next, we estimated the change in LD among these 2,670,445 polymorphic pairs after the 21 generations of random mating used to construct the SAIP. We binned the minor allele frequencies of the SAIP into 0.01-increments from 0 to 0.5. For each of the polymorphic pairs, we calculated the range of allele frequencies pb at variant B that would result in high LD  given a binned allele frequency pa at variant A (Methods) [It is quite likely that linkage disequilibrium (LD), the non-random segregation of allelic variants at two loci, affects our results. Estimating LD using pooled sequence data is a difficult challenge as the haplotypes of each fly are not known . Althougpulation . Moreovepulation , 87, andpulation , suggestpulation \u201390. Thispecified . We therMethods) . At gene2R  did not decrease in the short- or long-sleeper populations; instead, the average distance between SNPs in high LD tended to remain relatively constant with the exception of long-sleeper variants on chromosome 2R . Given tCVE with an FDR of 0.01 or less. In that study, night sleep duration and night sleep CVE were highly genetically correlated, and 95.6% of the polymorphisms identified for night sleep overlapped with night sleep CVE [Myb-interacting protein 120 (mip120) and scribbled (scrib), were implicated in night sleep duration in both studies, though only allele frequency changes in an intron of scrib exceeded the simulated drift threshold. Interestingly, 16 genes implicated in night sleep CVE in the GWAS overlapped the present study and included 5-hydroxytryptamine (serotonin) receptor 1A (5-HT1A), CG33158, CG34353, Dpr-interacting protein \u03b3, faint sausage, Fish-lips, frizzled (fz), Guanine nucleotide exchange factor in mesoderm, kin of irre (kirre), mip120, NK7.1, plum, scrib, still life, Synaptosomal-associated protein 25kDa, and Tie-like receptor tyrosine kinase. Of these genes, only CG34353, faint sausage, Fish-lips, fz, kirre, plum, and scrib had allele frequency changes that exceeded the simulated drift threshold. If all sleep traits measured in the GWAS were considered, 34 genes overlapped with the logistic regression\u201415 if the drift threshold was considered  of sleep using 167 lines of the DGRP . That stleep CVE . We founleep CVE , consistleep CVE \u201393 or arleep CVE  with GWAnsidered . Alternafz, shaggy (sgg), and dally are components of the Wnt signaling pathway [sgg is also part of the circadian rhythm pathway [pointed has functions in the dorso-ventral axis formation (Egfr) [scrib functions in the Hippo signaling pathway [skittles and small wing are part of the phosphatidylinositol signaling pathway. Furthermore, the propensity for selection for the minor allele in short sleepers and the major allele in long sleepers suggests that certain patterns of alleles might function in a single pathway. We used Flybase [CVE from the genome-wide association study of sleep using the DGRP [Of the 121 single nucleotide polymorphisms (SNPs) and 5 indels, 9 were in the 3\u2019-UTR region of 8 genes, 2 were in the 5\u2019-UTR of 2 genes, 11 were in the exon of 8 genes, 55 were in the intron of 46 genes, 17 were within 1 kb of 16 genes, and 32 were intergenic. Selection polymorphisms fell within the gene regions (\u00b1 1 kb of the coding region) of 80 candidate genes . We sear pathway , and sgg pathway ; pointedn (Egfr)  and MAPKn (Egfr)  pathways pathway ; and ski Flybase  to mine the DGRP  as well the DGRP , 101\u2013103the DGRP , 104\u2013116Minos element insertions in three candidate genes from the logistic regression with allele frequency differences between long and short-sleeping populations that exceeded the simulated drift threshold . We also tested three chromosomal deficiencies spanning candidate genes on chromosome 2R for their effects on night sleep. We discuss each of these tests in turn. CG33156 is a gene predicted to function as an NAD+ kinase due to its sequence similarity to yeast [Minos insertion in the exon region of CG33156 did not affect night sleep, but did reduce day sleep in male and female flies by 61 minutes on average (P <0.0001) (P = 0.0121), but no other effect on sleep  (P <0.0001) and reducing the latter by 6.7 bouts (P <0.0001) (sgg is a glycogen synthase kinase 3 with a known role in Drosophila circadian rhythms [Minos insertion in an exon of sgg slept 86 minutes longer during the night than controls on average (P <0.0001) (P = 0.0206)  . There won sleep . fz is w ligands . A Minos<0.0001) . This in<0.0001) . sgg is  rhythms . Male an<0.0001) . This in 0.0206) . In addiactivity . Thus, oMinos insertion lines. sgg is located on the X chromosome; thus, male progeny of crosses where the Minos sgg allele originates from male parent will not have the sgg Minos insertion. We therefore analyzed the effects of the trans-heterozygous genotypes on the sleep of males and females separately. We discuss here the effects of the trans-heterozygous genotypes on female sleep only, though all results are provided  . Though these mutations are in a genetic background distinct from that of the SAIP we used in the artificial selection protocol, we nevertheless observed effects on sleep not only in single-gene mutations, but epistatic and maternal effects among these candidate genes as well.To determine whether there were synergistic effects of these mutations on sleep, we tested full diallel crosses of the three provided  Tables. = 0.110) . ParentaMinos lines of 12 hours of night sleep, using the same protocol as for the selection populations. The sleep deprivation significantly affected night sleep in the three Minos lines and the control . However, flies deprived in the isogenic control line had little night sleep differences over time as compared to their non-sleep-deprived counterparts (PTreatment\u00d7Day = ns), suggesting that the stimulus was very mild (PTreatment\u00d7Day < 0.0001). sgg, fz, and CG33156 had reductions in night sleep on day 3 that were greater than that of the control line   ; Fig 12,ne sleep . The sgg24 hours . Likewisincrease . In addir values . Adding r values . In summ2R that included polymorphisms for night sleep identified in this study and night sleep and night sleep CVE in the GWAS (Df(2R)BSC273 decreased night sleep by 15 minutes relative to controls (P = 0.0195); Df(2R)BSC306 also decreased night sleep by 15 minutes relative to controls (P = 0.0336); and Df(2R)BSC274 increased sleep by 13.2 minutes relative to controls (P = 0.0257). Thus, the mapping did not reveal causal candidate genes in the region, but did suggest opposing effects on night sleep between two regions along the chromosome. One region extended from 13159579 bp to 13539889 bp and had decreased sleep, while the other extended from 13539889 bp to 13593272 bp and increased sleep. Further fine mapping is required to test each of the candidate genes in this region, as quantitative trait loci spanning large genomic regions are likely to fractionate into many smaller candidate regions [Finally, we used three deficiencies to map a 99.6-kb region of chromosome the GWAS . Deficie regions .Sh, sss, inc, and nAChR\u03b14 [Here we have used artificial selection to drive night sleep duration to extremely high and low levels. Our selection procedure also resulted in a positively correlated response to day sleep. This combined response produced mean 24-hour sleep times of 269 \u00b1 21.11 and 176.59 \u00b1 11.27 minutes in the short sleepers . This amount of sleep is comparable to short-sleeping flies with single mutations in  nAChR\u03b14 , 50, 51, nAChR\u03b14 , 120. Th nAChR\u03b14 . Third,  nAChR\u03b14 , 67. We  nAChR\u03b14 . However nAChR\u03b14 , 78, tho nAChR\u03b14 , 77, 78, nAChR\u03b14  may contIn long sleepers, mean 24-hour sleep times were 1073.25 \u00b1 14.84 and 1060.43 \u00b1 11.62 minutes . Only 40 flies (0.256%) had 720 minutes (12 hours) of night sleep. It seems unlikely that artificial selection could obtain 12 hours of mean night sleep, at least from the outbred starting population we created, because we were not able to increase the mean sleep above that of the longest-sleeping DGRP line. DGRP_335 averaged 688.76 minutes of night sleep , and nigfz mutants did not have increased sleep in the 24 hours after sleep deprivation either. There are several possible explanations for this pattern. First, the sleep homeostat may be disturbed in the selected populations. The disturbance is independent of the selection procedure since it was observed in all four selected populations. Alternatively, low or no rebound has been observed in genetically modified flies [One of the hallmarks of sleep as a homeostatic process is the recovery sleep experienced after sleep is disturbed. Both long- and short-sleeping populations responded to a mild mechanical sleep deprivation stimulus, exhibiting decreased sleep during the stimulation procedure and increasing their sleep immediately afterwards. However, there was not an increase in sleep over the subsequent 24-hour period. Interestingly, ed flies \u2013127, inded flies , 124. Peed flies , 128\u2013130ed flies \u201336, 125.ed flies  not meased flies , 133. Thed flies , 134. Wesgg, a glycogen synthase kinase 3\u03b2 (GSK-3\u03b2) that phosphorylates the canonical circadian clock gene timeless [sgg overexpression reduces circadian period, while its reduction increases circadian period in flies [sgg alleles exhibit a clinal distribution in North American flies, linking alleles of the gene with the forces of natural selection [sgg can alter sleep in flies in addition to its known function in circadian rhythms. Evidence from mouse studies suggests that GSK-3\u03b2 may also have a role in mammalian sleep. Mice with increased GSK-3\u03b2 enzymatic activity do not have changes in sleep duration, but instead have increased numbers of NREM and REM bouts as compared to wild-type mice [sgg may have a conserved role in sleep as well as circadian rhythms.General patterns of sleep changed in both long and short sleepers. Previous work has shown that night average bout length is positively correlated with night sleep duration, while night bout number is negatively correlated with night sleep duration , 71. Thutimeless . sgg ovein flies . sgg allelection . Here weype mice . In addiype mice . Thus, smip120 on chromosome 2R. Polymorphisms along a 99.6-kb region of chromosome 2R were implicated in either this study or the GWAS [arrow , crowded by cid, downstream of receptor kinase (MAPK pathway), mars, Vesicular monamine transporter (Vmat), CG13330, CG13331, and CG33156. Portions of this region are in LD in the DGRP [Vmat has already been shown to be relevant to sleep [3L were also estimated to be in high LD. Mapping at higher resolution will be required to determine the causal genes.Using the CMH test, we observed genome-wide allele frequency changes in the long and short sleeper populations across just 13 generations of selection, making it a challenge to pinpoint the true targets of selection. Remarkably, large numbers of changes also occurred in the control populations, which did not undergo any selection. We therefore assumed that any significant allele frequency changes in the control population were due to random genetic drift, laboratory adaptation, or potential adaptation to the admixture of genomes used to construct the SAIP, and removed them from further consideration. This does not mean that these polymorphisms are not relevant to sleep, however; they were simply indistinguishable from other potential causes in this experiment. Also, few polymorphisms with significant allele frequency changes via the CMH test were common to replicates within each selection scheme despite the similarities in night sleep duration phenotypes. Thus, different constellations of alleles may produce similar phenotypes, as previously observed in a comparison of replicate selection populations adapting to a novel environment ; alternathe GWAS  and inclthe DGRP , and Vmato sleep , 138. DeCVE), reflected by the slow decay of variability in sleep among individuals over time. In contrast, the variability in sleep among individuals from populations selected for long sleep decreased rapidly, greatly reducing CVE. This pattern was also observed in the GWA study [The allele frequency distribution of the significant polymorphisms identified for night sleep duration in the GWA study differs from the original distribution of these alleles in the DGRP. The average minor allele frequency of a night sleep duration polymorphism in that study was 0.088. At generation 0, these polymorphisms were far more common in the outbred population constructed in this experiment, with an average minor allele frequency of 0.232. Yet it was a different group of polymorphisms, also at a relatively high average minor allele frequency (0.337) in generation 0, that are the putative targets of artificial selection. Both groups of candidate polymorphisms have some similarities, however. Most of the candidate polymorphisms identified in the present study were selected in the direction of the minor allele for short-sleepers , and towWA study . One of WA study . Alternamip120 and scrib, were implicated in the GWAS for night sleep, and an additional 16 were associated with night sleep CVE [foraging and fz) [heimdall) [5-HT1A, foraging, Calreticulin, and Semaphorin 5C) [CG33156, fz, and sgg are candidate genes for night sleep using mutations. Our results measuring sleep in trans-heterozygous crosses among these mutations demonstrate that sleep can be altered by dominance, epistatic, and maternal effects. Future functional validation is needed to validate the role of the remaining genes in sleep and their relationship to each other in the network. If this network of highly conserved genes indeed represents a core sleep network, one might predict that allele frequency shifts in this network will 1) underlie pleiotropic responses to changes in environmental conditions [As previous experiments in olfaction, starvation resistance, startle response, chill coma recovery, and food intake have suggested, the genetic overlap of natural population-based studies is neither single nucleotides nor single genes, but instead occurs among genetic pathways , 91, 92.leep CVE . Mutatio and fz) , 139, sleimdall) , or 24-horin 5C) . Genes iorin 5C)  and rabborin 5C) . Wnt sigorin 5C) , and werorin 5C) . The MAPorin 5C)  and mammorin 5C) . Mutatioorin 5C) , 43, 73.nditions  and 2) bDrosophila Genetic Reference Panel (DGRP) [We constructed an outbred population of flies using ten lines from the l (DGRP) , 81 withl (DGRP) . The othl (DGRP) . All tenhttp://flystocks.bio.indiana.edu/Fly_Work/media-recipes/bloomfood.htm) in constant temperature (25\u00b0C), humidity (60%), and light:dark cycle (12hr:12hr). To measure sleep, we collected male and female virgins from each population. Virgins were maintained in single-sex vials of 20 flies for four days before the experiment began  to control for any potential effects on sleep of mating status [2 anesthesia or any other potential acclimation effects, the first day of data recording was discarded. Flies were visually examined after the sleep and activity recordings were completed; data from any flies that did not survive the recording period was discarded. Sleep Analysis v6-1 software  was used to calculate night and day sleep duration in minutes, night and day sleep bout number, and night and day average sleep bout length; it also calculated sleep latency, the time in minutes to the first sleep bout after lights are turned off and waking activity, the number of activity counts per minute spent awake. In addition, we calculated the coefficient of environmental variation (CVE) for each sleep trait as (\u03c3E/\u03bc)\u00d7100 [\u03c3E is the within-replicate population standard deviation, and \u03bc is the within-replicate mean. We used an in-house C# program  to partition the activity counts for each fly as asleep, awake, or in a transient pause (1 to 4 minutes of inactivity). We used this information to calculate the percentage of flies in each state over time.Flies were maintained and assayed on standard food \u00d7100 , where \u03c3At Generation 0 of the selection procedure, we divided the outbred population into six populations by seeding six bottles with 25 randomly chosen flies of each sex. Two populations served as replicate unselected controls (C1 and C2), two populations were selected for long night sleep (L1 and L2), and two populations were selected for short night sleep (S1 and S2). We measured sleep in 100 males and 100 females from each of these populations prior to initiating the artificial selection procedure. We calculated differences in sleep among these populations using an ANOVA model (see Quantitative genetics of selection below for the model). The resulting analyses indicated that there were no significant differences in any sleep trait among these populations, with the exception of night bout number . Thus, eWe implemented the following artificial selection procedure each generation. First, 100 virgins of each sex were collected from each population bottle and placed into the sleep monitors. Sleep and activity were monitored continuously over a five-day period. Second, we calculated all sleep parameters for every fly in each selection population. For the four selection populations , the 25 males and females with the most extreme (high or low) night sleep within each population were chosen as parents for the next generation of that population. For the control populations (C1 and C2), we chose 25 males and 25 females at random to be the parents for the next generation of each population. We repeated this procedure for 13 generations. After generation 13, the six populations were maintained by seeding culture bottles with 25 randomly chosen flies of each sex.Minos element lines (see below) of sleep. We used an in-house Python program to calculate the number of flies asleep during any given minute, and to compute the average amount asleep per 30 minutes. We examined the overall efficacy of the sleep deprivation protocol for each population/line separately using the ANOVA model Y = \u00b5 + T + D + S + T\u00d7D + T\u00d7S + D\u00d7S + T\u00d7D\u00d7S + \u03b5, where T is the control or sleep deprived treatment, D is day, and S is sex. To determine whether night and/or day sleep changed across days for the sleep-deprived group and their respective controls, we analyzed the data for each population/line separately using the reduced ANOVA model Y = \u03bc + D + S + D\u00d7S + \u03b5, where D and S are as defined above. Post-hoc Tukey analysis was used to determine which days had significant changes in sleep duration. In addition, to assess sleep deprivation in the Minos lines, we assessed differences between mutants and controls per day using the model Y = \u03bc + T + L + S + T\u00d7L + T\u00d7S + S\u00d7L + T\u00d7L\u00d7S + \u03b5, where T and S are as defined above and L is Line.We subjected artificially-selected flies to sleep deprivation at Generation 60 using the following procedure. Individual flies were loaded into the DAM2 Trikinetics monitors using the protocol above. After allowing the flies to acclimate in the monitors for one day, we recorded normal sleep and activity for two days. We deprived the flies of sleep on the night of the third day using a standard vortexer  with a custom mounting plate . The vortexer gently shook (speed = 2.0) the flies for five seconds every minute of the 12-hour night sleep period. We allowed the flies to recover for two additional days. An identical set of flies was placed into Trikinetics monitors on a different shelf in the same incubator to serve as a non-sleep-deprived control. We repeated these experiments two times. We used this protocol to deprive the long- (L1 and L2) and short-sleeper (S1 and S2) populations. We also applied this protocol to deprive the We evaluated two different life-history traits in the selected and control populations of flies. At generation 14, flies were assayed for lifespan. 100 male and female virgins of each population were placed into 10 replicate vials per sex per population, with 10 same-sex flies per vial. Every two days, the flies were tipped into vials containing fresh food, and deaths were recorded until all flies were dead. Flies were assayed for egg-to-adult viability for nine generations, beginning at generation 4 and continuing through generation 12. Two replicate fly cultures per population per generation were seeded with 25 flies of each sex. Parents were cleared from the vials after 3 days, and the numbers of adult flies emerging were counted from days 10\u201314.Y = \u03bc + Sel + Rep(Sel) + Sex + Gen + Sel\u00d7Sex + Sel\u00d7Gen + Rep(Sel)\u00d7Sex + Rep(Sel)\u00d7Gen + Sex\u00d7Gen + Sel\u00d7Sex\u00d7Gen + Rep(Sel)\u00d7Sex\u00d7Gen + \u03b5, where Y is night sleep duration, Sel is the selection procedure (Short or Long night sleep); Gen is the fixed effect of generation; Rep is the random effect of replicate population; and \u03b5 is the variance within populations [P < 0.05) Sel term indicates that the selection procedure was effective, and a significant Sel\u00d7Sex term reveals sex-specific responses to selection. A significant Sel\u00d7Gen (Sel\u00d7Sex\u00d7Gen) term implies that the response to selection differed across generations (and sexes). We used the same model to determine whether the other sleep parameters and egg-to-adult viability had a correlated response to selection for night sleep. To further characterize sex- and generation-specific responses to selection for night sleep, we used the reduced model Y = \u03bc + Sel + Rep(Sel) + Gen + Sel\u00d7Gen + Rep(Sel)\u00d7Gen + \u03b5 to evaluate sleep characteristics in each sex separately; and the reduced models Y = \u03bc + Sel + Rep(Sel) + Sex + Sel\u00d7Sex + Rep(Sel)\u00d7Sex + \u03b5 and Y = \u03bc + Sel + Rep(Sel) + \u03b5 to analyze the response to selection in each generation separately and for each generation in each sex separately, respectively. The model Y = \u03bc + Sel + Rep(Sel) + Sex + Sel\u00d7Sex + Rep(Sel)\u00d7Sex + \u03b5 was used for the lifespan analysis. All analyses were implemented using SAS . To assess the response of night sleep to selection, we used the breeder\u2019s equation \u03a3R = h2\u03a3S to calculate the realized heritability h2, where \u03a3R is the cumulated response to selection, calculated as the mean night sleep of the offspring minus the mean night sleep of the parental generation; and \u03a3S is the cumulated selection differential, calculated as the mean night sleep of the selected parents minus the mean night sleep of the parental generation [To evaluate the effectiveness of the selection procedure, we used the equation ulations . A signineration . The samFor each generation of selection, we flash-froze 35 flies of each sex from each of the selection and control populations. We extracted DNA from these flies using a cell lysis solution {1.58 g of Tris-HCl , 37.22 g EDTA disodium salt  and filled to 1 liter with RNase/DNase-free water, adjusting the pH to 8.0 with 10 M NaOH  when necessary}. Flies were homogenized in 300 \u03bcl of cell lysis solution and four to six 2.38 mm metal beads  using an Omni Bead Ruptor {5 cycles of 5 seconds ON/ 5 seconds OFF\u2014Setting 5 m/s, equal to 3100 rpm }. An additional 400 \u03bcl of cell lysis solution, 120 \u03bcl of 10% SDS , and 60 \u03bcl of Proteinase K  were added and the samples were incubated at 65\u00b0C for 1 hour. The lysate was then transferred to a clean 1.5 ml tube, leaving the beads behind. RNA was eliminated from the lysate by adding 3.5 \u03bcl RNase A (20 mg/ml) , mixing, and incubating at 37\u00b0C for 15 minutes. Protein precipitation was initiated by adding 200 \u03bcl of Ammonium Acetate  solution (289.1 g of Ammonium Acetate in 500 ml of RNase/DNase-free water) to tubes chilled on ice for 5 minutes. Samples were vortexed at high speed for 20 seconds to mix, incubated on ice for 15 minutes, and then centrifuged at maximum speed for 5 minutes. The supernatant was then transferred to a clean 1.5 ml tube, and 3 \u03bcl of glycogen  was added. 700 \u03bcl of 100% isopropanol  was added and mixed to precipitate the DNA; samples were incubated for 1 hour at -20\u00b0C. The samples were then centrifuged at maximum speed for 5 minutes. The supernatant was poured off and the DNA pellet was washed with 600 \u03bcl of 75% ethanol , then centrifuged at maximum speed for 5 minutes. The ethanol was poured off and the pellet air-dried for 15 minutes. Samples were re-hydrated in 120 \u03bcl of RNase/DNase-free water.DNA samples were then purified using a phenol-chloroform extraction. First, 80 \u03bcl of 10 mM Tris , 1 mM EDTA, pH 7.8 and 200 \u03bcl of fresh phenol:chloroform:isoamyl alcohol (25:24:1)  were added to the DNA sample. The samples were vortexed for 30 seconds, then centrifuged at maximum speed in a 4\u00b0C centrifuge for 5 minutes. The resulting upper layer (~ 170 \u03bcl) was transferred to a fresh 1.5-ml tube. An equal volume of chloroform  (200 \u03bcl) was added. The sample was vortexed and centrifuged at maximum speed in a 4\u00b0C centrifuge for 5 minutes. The resulting upper layer (~ 150 \u03bcl) was transferred to a fresh 1.5-ml tube. Next, 20 \u03bcl of sodium acetate (NaOAc) , 500 \u03bcl of pure ethanol, and 1 \u03bcl glycogen were added. The sample was mixed and placed in ice for 15 minutes. Afterwards, the samples were centrifuged at maximum speed in a 4\u00b0C centrifuge for 30 minutes. The supernatant was removed, and the pellet washed with 0.5 ml of 70% ethanol. The sample was centrifuged at maximum speed for 5 minutes. The supernatant was removed and the sample air-dried for 5 minutes. The resulting DNA pellet was dissolved in 25 \u03bcl sterile 10 mM Tris, 0.1 mM EDTA, pH 7.8 and heated for 2 minutes at 55\u00b0C. DNA quantity was measured and quality evaluated (260/280 ratios of 1.8 or greater) using the Nanodrop 8000 .We sequenced DNA from each selection population and sex for the following generations: 0, 1, 2, 5, 8, 10, and 12. One microgram of genomic DNA was sheared to ~350 bp using a Covaris E210  with settings: duty cycle 10%; intensity 5; cycles/burst 200; time 45s. Libraries were constructed using the Tru-Seq DNA PCR-Free LT Sample Prep Kit  according to the manufacturer\u2019s protocol. The libraries were pooled in groups of 24 and run on a HiSeq2500  with version 3 sequencing reagents to generate a minimum of 37 million paired-end 126 base reads per library. The HiSeq data was processed using RTA1.18.64 and CASAVA 1.8.2.D. melanogaster assembly BDGP Release 6, UCSC version dm6 using with Burrows-Wheeler Alignment-MEM version 0.7.12 [http://github.com/nhansen/BardCNV) with the option -minqual 20 to filter reads for a minimum phred quality of 20. Counts for reads spanning indels were performed by first widening indel variants to their narrowest unambiguous region, then tallying spanning reads both with and without the indel using the perl module Bio::SamTools (http://search.cpan.org/~lds/Bio-SamTools/lib/Bio/DB/Sam.pm). Confidence interval boundaries with the highest posterior density for the estimated read allele proportions were calculated in R using CRAN \u2018binom\u2019 package\u2019s \u2018binom.bayes\u2019 function. For downstream analyses, we considered all bialleleic polymorphisms with total coverage of 10 and less than 2000 in each population [Sequences were aligned and mapped to the n 0.7.12  and Novon 0.7.12 , after cn 0.7.12 . Reads wn 0.7.12 . Duplican 0.7.12  with defpulation .P-value (2.3 \u00d7 10\u22128) as the threshold of significance for the CMH tests in order to account for multiple tests. We made this calculation for both sexes combined as sleep in both sexes responded to the selection procedure , Gen is generation, and Sel is selection scheme (Long or Short). Both long and short selection population replicates for all seven generations and both sexes were put into the logistic regression analysis. We required that the P-value for the lack of fit be \u2265 0.05 ; the P-value for Sel to be \u2264 0.05, and the P-value for Gen\u00d7Sel to be \u2264 0.05 [We used the Cochran-Mantel-Haenszel (CMH) Test to detect changes in allele frequency for all known segregating 2,222,264 and putative de novo 258,268 polymorphisms  between e \u2264 0.05 . All anapa, in 0.01 increments from 0.01 to 0.5. We then calculated the lower and upper bounds of the corresponding minor allele frequency at SNP B, pb, that would result in an r2 of 0.8 or greater using the following formulations from VanLiere et al. [We chose 3,600 SNPs at random per chromosome arm and calculated linkage disequilibrium among these SNPs for the 10 DGRP lines using plink 1.0.7 . To estie et al. :pb is r2pa/(1 + r2pa\u2212pa).The lower bound of pb is the minimum of pa/(r2 \u2013r2pa + pa), or 0.5.The upper bound of r2 \u2265 0.8) with SNP A.We determined whether the actual minor allele frequency at SNP B was within the upper and lower bounds given by these two equations. If it was, we assumed that SNP B was in high LD  to estimate recombination rates for the 126 variants we identified using logistic regression. Estimates based on Marey map equations [In addition, we used the quations  and on equations  are inclNe, of our populations ast is the time in generations, q0 is the starting allele frequency at generation 0, and \u03c32 is the variance in allele frequencies [Ne using the equation above for each allele frequency bin at Generation 12. The median Ne was 42.5  which we applied to the drift simulations. Drift was simulated for a single SNP by random and independent sampling of parental genotypes over 12 generations for a given starting minor allele frequency [X chromosome, to represent the long-sleeper and short-sleeper populations with replication. We then calculated the absolute value of the average difference between short and long sleeper populations for each starting minor allele frequency. The 99.9% quantile was used as an upper bound of the potential allele frequency change due to drift for each starting minor allele frequency (65).We compared the actual allele frequency changes in the control populations with simulated random genetic drift. We first estimated the variance effective population size, quencies . Using tquencies . We estirequency . We simuMinos element Mi{ET1} insertion lines [CG33156, fz, and sgg. The alleles tested were CG33156MB05931, fzMB07478, and sggMB03827 . The Mi{ET1}CG33156MB05931 insertion is located on chromosome 2R at position 13,549,134, within an exon common to all 5 isoforms of CG33156. The Mi{ET1}fzMB07478 insertion is located at position 14,303,880 on chromosome 3L in the second intron of both isoforms of fz. The Mi{ET1}sggMB03827 insertion is located at position 2,673,583 on the X chromosome; this insertion is in an exon common to all 17 isoforms of sgg. These insertions were created in an isogenic w1118 background (w1118{5905}); we used this background as a control [Y = \u03bc + G + S + R + G\u00d7S + G\u00d7R + R\u00d7S + G\u00d7S\u00d7R + \u03b5, where G is genotype, S is sex, R is replicate, and \u03b5 is the error term. Males and females were analyzed separately using the reduced ANOVA model Y = \u03bc + G + R + G\u00d7R + \u03b5.We tested on lines  in threeMinos lines in a full diallel design, so that all possible trans-heterozygous combinations were represented. Five virgin females of each line were crossed to five males of each line. sgg is located on the X chromosome; thus, male progeny from male sgg parents did not have the sgg mutation. We therefore restricted our analysis to males and females separately. We compared differences in sleep across trans-heterozygous genotypes using the ANOVA model Y = \u03bc + G + R + G\u00d7R + \u03b5 as defined above. Genotypic effects can be decomposed into general combining ability (GCA), specific combining ability (SCA) and reciprocal effect (REC) using the full diallel, fixed effects design  of Griffing [Yij, where Y is the mean of the ijth genotype. We estimated the GCA of a mutation as (1/2n)(Xi. + X.j)-(1/n2)X.., where Xi. and X.j are the sums of mean sleep phenotypes of heterozygotes with the ith and jth Minos element, respectively; X.. is the sum of mean sleep phenotypes for all genotypes; and n equals the number of mutations (3 in this case) [Yij + Yji)\u2013(1/2n)(Xi. + X.i + Xj. + X.j) + (1/n2)X.. [Yij\u2212Yji) [Y = \u03bc + GCA + SCA + REC + GCA\u00d7R + SCA\u00d7R + REC\u00d7R +\u03b5, where \u03bc is the overall mean and \u03b5 is the error term [We also investigated potential interactions among these candidate genes by crossing them together. We crossed the three Griffing . The GCAGriffing , 118. Meis case) . We esti1/n2)X.. . REC effYij\u2212Yji) . The GCAYij\u2212Yji) , 158. Weror term . We usedror term .Mi{ET1} lines were available for mip120, the candidate gene common to this study and the genome-wide association of sleep. However, many candidate genes were located along a 99.6-kb region on chromosome 2R, including crowded by cid, mip120 and CG33156 from the present study, and arrow, downstream of receptor kinase, mars, mip120, Vmat, CG13330, and CG13331 from the genome-wide association study [Df(2R)BSC273, Df(2R)BSC274, and Df(2R)BSC306, which have breakpoints from 13159579\u201313502150, 13430464\u201313593272, and 13364133\u201313539889, respectively. These deletions were created in an isogenic w1118 background (w1118 {6326}); we used these backgrounds as a control [CyO balancer chromosome. We crossed 5 virgin females of each deficiency line to 5 males of the w1118 {6326} control and compared the heterozygotes bearing the deficiency to the control line. Sleep phenotypes were measured as described above for 16 flies/sex/genotype, and the measures were repeated two times. Sleep phenotypes were analyzed using the ANOVA model Y = \u03bc + G + S + G\u00d7S + R(G\u00d7S) + \u03b5, where G is deficiency genotype, S is sex, R is replicate, and \u03b5 is the error term. All stocks were obtained from the Bloomington, Indiana stock center. All analyses were implemented using SAS .No on study . We theron study  Df(2R)BS control . The chrData availability: Raw DNA sequence data have been deposited in the NCBI SRA database under BioProject PRJNA369048, SRA database SRP098556.S1 FigThe graphs show the differences among the sleep phenotypes in the control, long, and short sleep populations at Generation 0, prior to artificial selection. Mean \u00b1 SE are plotted for sexes combined. C1 and C2, control population replicates 1 and 2; L1 and L2, long-sleeping population replicates 1 and 2; S1 and S2, short-sleeping populations 1, and 2. (A), night sleep; (B), Day sleep; (C), night bout number; (D), day bout number; (E), night avg. bout length; (F), day avg. bout length; (G), sleep latency; (H), waking activity.(PPTX)Click here for additional data file.S2 FigCVE; (C) day average bout length; (D) day average bout length CVE; (E), waking activity; and (F), waking activity CVE. , Mean \u00b1 SE are plotted for sexes combined. , mean is plotted for sexes combined. Light blue and dark blue triangles indicate Replicate 1 and Replicate 2 populations selected for long sleep; Light red and dark red squares indicate Replicate 1 and Replicate 2 populations selected for short sleep; and light gray and black circles indicate Replicate 1 and Replicate 2 control populations.(A), night bout number; (B), night bout number (PPTX)Click here for additional data file.S3 FigShown are the percentages of flies sleeping, awake, or in a transient pause for each minute in a 24-hour day. (A), control females; (B), control males; (C), long sleeper females; (D), long sleeper males; (E), short sleeper females; (F), short sleeper males. Black/dark blue/dark red = sleep; gray = transient pause; light gray/light blue/light red = wake. White and black bars on the x-axis indicate the light and dark periods, respectively.(PPTX)Click here for additional data file.S4 Fig(A), the difference in 24-hour sleep from baseline (the average of days 1 and 2) sleep is plotted for day 3 (dark blue bars) and for day 4 (light blue bars). (B), the difference in day sleep from baseline for day 4.(PPTX)Click here for additional data file.S5 Fig2L; (B), chromosome 2R; (C), chromosome 3L; (D) chromosome 3R; (E) chromosome X; (F) all insertions and deletions.(A), chromosome (PPTX)Click here for additional data file.S6 FigP <0.05; ***, P <0.001; ****, P <0.0001.(A), day average bout length; (B), night bout number; (C), waking activity. *, (PPTX)Click here for additional data file.S7 FigP < 0.05; P-values reflect the comparison of deprived and/or recovery day sleep with baseline sleep.(A), the difference in 24-hour sleep from baseline (the average of days 1 and 2) sleep is plotted for day 3 (dark blue bars) and for day 4 (light blue bars). (B), the difference in day sleep from baseline for day 4. * (PPTX)Click here for additional data file.S8 Figr2 values estimated to be below 0.8, and blue for r2 values \u2265 0.8.For each population and generation, the LD is plotted as yellow for (PPTX)Click here for additional data file.S1 Tabled.f., degrees of freedom; M.S., Type III mean squares; F, F ratio statistic; P, P-value.For each sleep trait, the ANOVA results are presented. gen, generation; rep, replicate; sel, long or short selection scheme; (XLSX)Click here for additional data file.S2 Tabled.f., degrees of freedom; M.S., Type III mean squares; F, F ratio statistic; P, P-value.For each sleep trait listed, the ANOVA results are presented. rep, replicate; sel, long or short selection scheme; (XLSX)Click here for additional data file.S3 Tabled.f., degrees of freedom; M. S., Type III mean squares; F, F ratio statistic; P, P-value.For each sleep trait listed, the ANOVA results are presented. rep, replicate; (XLSX)Click here for additional data file.S4 TableCVE trait listed, the ANOVA results are presented. d.f., degrees of freedom; M.S., Type III mean squares; F, F ratio statistic; P, P-value.For each sleep (XLSX)Click here for additional data file.S5 Tabled.f., degrees of freedom; MS, Type III mean squares; F, F ratio statistic; P, P-value.rep, replicate; sel, long, short, or control selection scheme; (XLSX)Click here for additional data file.S6 TableP-values for the generation, selection scheme (sel), generation\u00d7sel, and the lack-of-fit statistic.Listed are the chromosome, position in base pairs, the selection population , selection scheme (long or short), sex, replicate, minor allele frequency for each generation, which generation(s) the polymorphism was significant for the CMH test, whether the polymorphism is located in an inversion region, and the (XLSX)Click here for additional data file.S7 Tableg and as mapped by Comeron et al.h are given. Genes previously identified as candidate genes for night sleep or night sleep CVE from the genome-wide association study of the DGRP are indicated. Genes previously implicated in other sleep or circadian rhythm studies are indicated. Polymorphisms falling within two or more genes are included with a separate row for each gene.Each polymorphism is listed by chromosome, position in base pairs, and type of polymorphism (SNP or indel). Variants whose average allele frequency difference between the long- and short-sleepers exceeded the largest change expected due to drift are indicated, as are the putative LD blocks for long-and short-sleeper populations. If the polymorphism was within a gene region or \u00b1 1kb from a gene region, the Flybase ID, gene symbol, and polymorphism location are given. The midpoint recombination rates (RRC) as calculated by Fiston-Lavier et al.(XLSX)Click here for additional data file.S8 TableListed are the gene symbol and the number of polymorphisms per gene (if any were found) for each trait that were identified in the GWAS.(XLSX)Click here for additional data file.S9 TableCandidate genes with known physical and genetic (enhanceable or suppressible) interactions are indicated. In some cases, the candidate sleep gene is the source; in others, it is the target. This is indicated by the Source gene and Target gene columns. Also listed are Flybase ID numbers, whether the genes were candidate genes from a genome-wide association study, previously identified sleep genes, or previously identified circadian genes. Genes without highlights have average allele frequency differences greater than that predicted by drift simulation; highlighted genes did not pass this threshold difference. Publications supporting the known physical and genetic interactions among genes are given as PubMed ID\u2019s and are listed in (XLSX)Click here for additional data file.S10 TablePublications are numbered according to the gene entries in (DOCX)Click here for additional data file.S11 TableP values reflect the comparison of the Minos element in the gene listed versus the isogenic control. rep, replicate; d.f., degrees of freedom; M.S., Type III mean squares; F, F ratio statistic; P, P-value. Significant comparisons are shown in bold.For each sleep trait, the ANOVA results are presented. (XLSX)Click here for additional data file.S12 TableP values reflect the comparison among all trans-heterozygous crosses. rep, replicate; d.f., degrees of freedom; M.S., Type III mean squares; F, F ratio statistic; P, P-value. Significant comparisons are shown in bold.For each sleep trait, the ANOVA results are presented. (XLSX)Click here for additional data file.S13 TableP values reflect the comparison among all trans-heterozygous crosses. rep, replicate; d.f., degrees of freedom; M.S., Type III mean squares; F, F ratio statistic; P, P-value. Significant comparisons are shown in bold.For each sleep trait, the ANOVA results are presented. (XLSX)Click here for additional data file.S14 Tablet-test statistic and corresponding P value indicate the degree of significance of the GCA effect. Significant comparisons are shown in bold.For each sleep trait, the estimate of general combining ability (GCA) is listed, along with the standard error. The (XLSX)Click here for additional data file.S15 Tablet-test statistic and corresponding P value indicate the degree of significance of the SCA effect. Significant comparisons are shown in bold.For each sleep trait, the estimate of specific combining ability (SCA) is listed, along with the standard error. The (XLSX)Click here for additional data file.S16 Tablet-test statistic and corresponding P value indicate the degree of significance of the REC effect. Significant comparisons are shown in bold.For each sleep trait, the estimated reciprocal effect (REC) is listed, along with the standard error. The (XLSX)Click here for additional data file.S17 TableFor each sleep trait, the genotype of the male, female, and progeny are listed, along with the number of flies tested, the mean phenotype, and the standard error.(XLSX)Click here for additional data file.S18 TableP values reflect the comparison of the deficiency listed versus the isogenic control. rep, replicate; d.f., degrees of freedom; M.S., Type III mean squares; F, F ratio statistic; P, P-value. Significant comparisons are shown in bold.For each sleep trait, the ANOVA results are presented. (XLSX)Click here for additional data file."}
+{"text": "Patient derived xenografts (PDX) are generated by transplanting the original patient\u2019s tumor tissue into immune-deficient mice. Unlike xenograft models derived from cell lines, PDX models can better preserve the histopathology from the original patient and molecular pathways. High-grade serous carcinoma (HGSC) is a deadly form of ovarian/fallopian tube cancer whose response to current chemotherapies varies widely due to patient variability. Therefore, a PDX model can provide a valuable tool to study and test treatment options for each individual patient.In this study, over 200 PDX tumors from nine HGSC were analyzed to investigate the nature and behavior of PDX tumors originating from HGSC. PDX tumors were serially passaged (from P0 to P4) and tumors were grafted orthotopically under the ovarian bursa or subcutaneously.Comparative analysis of the histology and molecular markers of tumors from over 200 PDX tumor-bearing mice, revealed that the tumors maintained similar histologies, stem cell populations, and expression for the majority of the tested oncogenic markers, compared to the primary tumors. However, a significant loss of steroid hormone receptors and altered expression of immunoresponsive genes in PDX tumors were also noted. Our findings provide substantial new information about PDX tumor characteristics from HGSC which will be valuable towards the development of personalized therapy and new drug development for HGSC.The online version of this article (doi:10.1186/s13045-016-0318-6) contains supplementary material, which is available to authorized users. Epithelial ovarian cancer (EOC) has a disproportionately high mortality rate in comparison to other female malignancies . AccordiTraditionally, the assessment of experimental cancer therapies using the established ovarian cancer cell lines have many limitations and cannot truly reflect the complexity and interpatient variation of ovarian cancer. Patient-derived xenograft (PDX) or a xenopatient is a system in which a portion of a patient\u2019s tumor, obtained either by surgical resection or biopsy, is transplanted in immunodeficient mice and allowed to propagate without any in vitro manipulation. Tumors can be engrafted heterotopically or orthotopically , and botEOC tumors are highly heterogeneous, with variable responses to standard chemotherapies emphasizing the need for PDX models to study EOC diversity and aid in novel therapeutical development . It is aFresh primary ovarian carcinoma tissues were obtained from chemotherapy na\u00efve ovarian cancer after resection at the Prentice Women\u2019s Hospital of Northwestern University from September 2013 to June 2014. Prior to surgery, written informed consent for tissue acquisition was obtained and nine consecutive cases of HGSC were collected. All tumors were collected and engrafted within 2\u00a0h post resection. Normal fallopian tube tissues were collected as normal control. Each case was reviewed by pathologists to confirm the diagnosis. The collection of human tissue specimens and the PDX mouse protocol were approved by the Institutional Review Board and Institutional Animal Care and Use Committee at Northwestern University. The clinical and pathological features of patients are summarized in Table\u00a0Total RNA was isolated using the Trizol reagent (Invitrogen) and PureLink RNA Mini Kit (Ambion) according to manufacturer\u2019s instructions. RNA quantity was assessed by NanoDrop 1000 spectrophotometer, Agilent 2100 bioanalyzer, and PCR bioanalysis and samples with an RNA integrity number (RIN) that scored higher than 8.0 were used. Expression profiling was performed using a HumanHT-12 v4 Expression Beadchip (Illumina) at the Northwestern Genomic Core Facility. Expression data were normalized using the median normalization. After normalization, significant differentially expressed mRNAs were identified through volcano plot filtering. Finally, hierarchical clustering was performed to show distinguishable mRNA expression profiling among samples.The genomic DNA of nine primary cases was extracted and purified using the QIAamp DNA FFPE Tissue Kit (QIAGEN) according to the manual. P53 exon4-9 mutation analysis was conducted as previously described . In brieRNA isolation and quantitative real-time PCR (qPCR) was conducted similarly as before . Brieflynull or NSG mice (The Jackson Laboratory) were used. Mice were maintained in laminar flow rooms, maintaining consistent temperature and humidity and were given free access to water and a normal diet. Mice were housed for 14\u00a0h light and 10\u00a0h dark cycle. Experiments were approved by the Institutional Animal Care and Use Committee of Northwestern University. Xenografted tissues were labeled as passage 0 (P0), P1, P2, etc. depending on the number of passages from the initial tumor.Eight-to-twelve-week-old female adult non-obese diabetic (NOD)-scid IL2R\u03b3For the first generation (P1) of xenografted, fresh tumor tissues (P0) collected from patients were cut into small (~3\u2009\u00d7\u20093\u2009\u00d7\u20092\u00a0mm) fragments, and then two tissue fragments were subcutaneously xenografted to each dorsal? flank of a NSG mouse.For other generations (\u2265P2), tumor tissues were cut into small pieces (~2\u2009\u00d7\u20092\u2009\u00d7\u20092\u00a0mm). Then, two to four tissue fragments were subcutaneously xenografted into two dorsal? flanks of NSG mice. First, mice were anesthetized by intraperitoneal injection of ketamine/xylazine (90/8\u00a0mg/kg), and the mice were shaved on the back where the surgery would occur, and the site was disinfected with providone iodine prep pads and alcohol swab . An one cm in length incision was made in the skin at the midline of the mouse back, and four separate tumor fragments were put into the upper left, upper right, lower left, and lower right of back, accordingly. After implantation, the skin was sutured, and mice were revived.Tumor tissues were cut into small pieces (~1\u2009\u00d7\u20091\u2009\u00d7\u20091\u00a0mm) and grafted onto the left side of the ovarian intrabursa of adult female NSG mouse hosts. The procedure of implantation for IB xenograft is the same as previously described . Mice weMice were sacrificed when the tumor size reached 1.5\u00a0cm in diameter or ascites emerged. Body weight was measured, and mice were sacrificed by intraperitoneal injection of ketamine/xylazine (90/8\u00a0mg/kg).a\u2009\u00d7\u2009b\u2009\u00d7\u2009c\u2009\u00d7\u2009\u043b/6, where a is the length, b is the width, and c is the height. All organs in the peritoneal, pelvic, thoracic, and cranial cavities were dissected out and checked for possible metastasis. Numbers of metastasis was documented and images of metastasis were photographed. Female reproductive tissues including the bilateral ovaries and uterus were isolated and fixed in modified Davidson\u2019s fixative. The other organs including brain, heart, lungs, liver, pancreas, spleen, kidneys, stomach, intestine, cecum, rectum, omentum, and diaphragm were also collected and fixed in modified Davidson\u2019s fixative. All fixed tissues were processed, embedded in paraffin, and sectioned and then hematoxylin and eosin (H&E) staining was performed for histologic examination.After dissection of the tumors, tumor size was documented by measuring tumor diameters. Then, tumor volume was calculated according to the formula TV (mm3)\u2009=\u2009a\u2009\u00d7\u2009b2\u2009\u00d7\u2009\u03c0/6, where a is the longest diameter, and b is the shortest diameter. Mouse was euthanized when a tumor reached 1.5\u00a0cm in diameter.Four tumor fragments were subcutaneously xenografted into two mice. Tumor growth was monitored by measuring tumor diameter every 2\u00a0weeks. Tumor volume was calculated according to the formula TV (mm3)\u2009=\u2009Tissue cores were collected from tumors for tissue microarray and represented in duplicate. Tissue microarrays were sectioned at 4\u00a0\u03bcm in thickness. Tissue microarray slides were deparaffinized in xylene and rehydrated in a graded series of ethanol. After antigen retrieval, all immunohistochemical staining was performed on a Ventana Nexus automated system. In brief, endogenous peroxidase activity was blocked with 3\u00a0% hydrogen peroxide. After blocking in 1.5\u00a0% normal goat serum for 30\u00a0min at room temperature, slides were then incubated overnight at 4\u00a0\u00b0C with primary antibodies in a humid chamber. Staining was detected with I-View 3,3\u2032-diaminobenzidine (DAB) detection system.Semiquantative immunointensity was scored as 0 (negative), 1 (weak), 2 (moderate) and 3 (strong) and percentage was showed as %. Immunoreactivity for HMGA2, MTSS1 and P16 was scored for intensity only. Immunoreactivity for Ki67, P53, P21, ER, PR, ALDH1, CD24, and CD133 was scored for percentage only. Antibodies used for this study were listed in Additional file t test and one-way ANOVA analysis were used to determine significance. P\u2009<\u20090.05 was considered statistically significant.The software SPSS V20.0 was used for statistical analysis. All data were presented as means and standard errors. Student\u2019s In this study, we designed a standard for workflow to establish PDX for HGSC and to evaluate the PDX tumor biology Fig.\u00a0. A frozeIB implantation of epithelial ovarian cancer has been performed successfully in our lab using ovarian cancer cell lines . The burThe success of engraftment (SOE) for primary ovarian cancer has not been defined. Based on a recent study of 241 cases, 12\u00a0months was the cutoff and an overall 74\u00a0% engraft rate was observed . Our curAll P0 to P4 HGSC were collected and subjected to histologic evaluation Fig.\u00a0. The mitTo evaluate the molecular differences of primary and engrafted tumors, we prepared a TMA to include all engrafted tumors 124 tumors) generated from eight cases   Fig.\u00a0. PathwayAttempts for PDX in ovarian cancer have existed for decades. In 1977, Davy et al. was the first to conduct subcutaneous (SQ) heterotransplants of ovarian cancer tissue into nude mice . In 1984IP and orthotopic models can mimic the patients of the metastasis pattern or ascites formation , 34. ThePDX of ovarian cancer can be passaged and retransplanted for up to six generations , 20, 36,One essential determinate of the validity of the PDX tumors is the maintenance of similar histologic and molecular characteristics of the repeated passages of PDX tumors to primary tumors. Several studies suggested that ovarian cancer PDX can maintain similar architectures and growth pattern as primary tumors , 37, 38,The published data suggested that both primary and PDX tumors maintained a similar molecular expression pattern , 38. To Proliferation and mitotic index seem to be higher in PDX tumors than primary ones . We founIt seems that current chemotherapies used in the clinic may be as effective in treating PDX tumors as primary ovarian cancer , 22. ForThere are several published data on ovarian cancer PDX models, but the results vary widely among studies due to different histologic subtypes, implantation site, and passage and analysis platforms. To use PDX as a tool for potential therapeutical purposes, it is very important to compare the histologic and molecular difference, stem cell change, and growth behavior in different microenvironments between primary and engrafting tumors. Therefore, an in-depth analysis of PDX tumors should include the engrafting site, passage time and level, microenvironment, and primary and metastatic tumors. To this end, we compared five of the most recent and similar studies and the results are summarized in Table\u00a0In summary, we established the heterotopic and orthotopic PDX for HGSC in this study. The histological and molecular analysis provided valuable information for the future use of HGSC PDX. Our findings support the complexity of ovarian tumor histology, stem cells, and molecular characteristics, indicating a need for a PDX model in order to develop personalized medical treatments for this deadly disease."}
+{"text": "Inorganic pollutants in milk and beef are of major public health concern; however, information in Africa is still limited due to low food safety monitoring practices. In this study, we established levels of lead (Pb), zinc (Zn), cadmium (Cd), copper (Cu), and iron (Fe) in milk and beef and obtained the estimated daily intake (EDI) and incremental lifetime cancer risk (ILCR) as measures of risk to the Ugandan population.  This was a cross-sectional study in which a total of 40 samples of milk and beef were collected from Bushenyi district in southwestern Uganda. Samples were analyzed by atomic absorbance spectrophotometer, and the EDI and ILCR were computed using the US EPA reference values. P < 0.05) Pb and Fe concentrations than milk. The EDI was highest in children, and this was followed by very high ILCR levels, showing that milk and beef are not safe for children in Uganda. Bearing in mind that a high HI was shown, beef and milk from these regions are not recommended for consumption especially by children although more studies remain to be conducted.  Heavy metal concentrations were highest in the order of Zn\u2009>\u2009Fe\u2009>\u2009Pb\u2009>\u2009Cu in milk samples, while in beef samples, concentrations were highest in the order of Zn\u2009>\u2009Pb\u2009>\u2009Fe\u2009>\u2009Cu and no Cd was detected. Furthermore, beef had significantly higher ( Heavy metals in milk and beef of Uganda may predispose the indigenous community to cancer and other health-related illnesses, showing a need for improved food safety screening to promote food safety. Animal food product contamination with inorganic pollutants has increased due to intensified human activities and industrialization . These iIn Uganda, recent evidence has shown that roasted beef sold in Central Uganda has high levels of inorganic pollutants , 12. ThiIn Uganda, Pb, Zn, and Cd continue to be key components of major agricultural products , and theFood safety studies on milk and beef in Uganda had previously placed a lot of emphasis on microbial load , 28, leaThis was a cross-sectional study in which milk and beef samples were collected from Bushenyi district. Bushenyi district lies in the cattle corridor of Uganda, and historically, it is associated with high milk and beef contributions to the Ugandan economy , 14. NamA total of 20 samples each of milk and beef were collected from 5 randomly chosen subcounties in Bushenyi district. In brief, samples were collected using sterile bottles for both milk (approximately 10\u2009ml) and beef (approximately 200\u2009g), transported under ice, and stored in a refrigerator at \u221220\u00b0C at the Department of Physiology, Faculty of Biomedical Sciences of Kampala International University Western Campus. These were subsequently transported frozen under ice to the Industrial Chemistry Laboratory in the School of Natural Sciences under Makerere University for Pb, Zn, Cd, Cu, and Fe analysis.Sample preparation and analysis were conducted using standard methods . In briey)\u2009=\u20090.0092\u2009\u00d7\u2009concentration (x), R2\u2009=\u20090.9784\u2009 Equation for Pb: absorbance . The stock solutions for Pb were acquired from E. Merck, D-6100 Darmstadt, FR Germany. Analysis was done using AAS, and the respective absorbance for each metal was read and a standard calibration curve was generated for each pollutant. From the standard curves, equations were generated and used to determine the concentrations for the samples as shown below:The homogenized samples were subjected to heavy metal analysis against Pb, Zn, Cd, Cu, and Fe. The absorbance for each sample was taken and the concentration determined in ppm using the equation generated from the standard curves.This was measured by using guidelines from the US EPA . EstimatC\u2009\u00d7\u2009IR)/BW, where C\u2009=\u2009concentration of the metal (mg/kg), IR\u2009=\u2009ingestion rate for beef, and BW=beef weight. The ingestion rate of 120\u2009g/day among adults and 56.7\u2009g/day among children in Uganda was used [EDI\u2009=\u2009ILCR=ectively .This was done using the following equation:e US EPA  was 0.00t-test was conducted for comparisons on concentrations among adults and children for each metal with P < 0.05 taken as significant. EDI levels were determined for adults and children and compared to the tolerable allowable intake levels (TAL). ILCR for allowable and nonallowable levels were indicated by superscripts \u201ca\u201d and \u201cb,\u201d respectively, after comparing them to the US EPA reference values in the normal range of 10\u22126 to 10\u22124 [Data were entered in duplicates and transferred into Microsoft Excel version 2013 for analysis. Information was presented descriptively as mean\u2009\u00b1\u2009SEM, and a two-sample  to 10\u22124 , 36. Fin to 10\u22124 .P > 0.05). In addition, significant differences (P < 0.05) were found to exist between Pb and Fe concentrations. In particular, Pb concentrations in beef were 18.90\u2009\u00b1\u20092.40\u2009ppm, while in milk, it was 10.48\u2009\u00b1\u20091.82\u2009ppm. Also, Fe levels were higher in beef than milk, that is, 17.04\u2009\u00b1\u20091.15 and 11.96\u2009\u00b1\u20091.00\u2009ppm as shown in The heavy metal concentrations were generally in the order of Zn\u2009>\u2009Fe\u2009>\u2009Pb\u2009>\u2009Cu in milk samples, while in beef samples, concentrations were highest in the order of Zn\u2009>\u2009Pb\u2009>\u2009Fe\u2009>\u2009Cu, and no Cd was detected in both milk and beef samples. Mean concentrations of Zn and Cu in both the milk and beef samples were not significantly different (P < 0.05) in the EDIs for milk were found to exist between adults and children as shown in The estimated daily intake (EDI) was generally all below the tolerable allowable levels except for lead in both adult and children. Furthermore, the EDI of zinc was highest in milk, especially in children, while that of copper in adults was the lowest. Furthermore, significant differences (P < 0.05) were found in ILCR levels in Zn within the adult and children population consuming beef while in milk, significant differences exist in ILCR levels for Pb and Zn as shown in The study showed that the incremental lifetime cancer risk (ILCR) was highest in beef, in both adults and children, primarily as a result of Pb levels. The ILCR was found dangerously high in milk for the children population due to high Pb levels. No significant differences in Pb ILCR levels were found in both adults and children for beef. Furthermore, significant differences (P < 0.05) in both adults and children. Very high Pb THQ led to elevated HI in beef. Also, THQ in milk was found to be significantly different in Cu and Zn (P < 0.05) in adults and children, and the HI in milk was found to be lower than that in beef as shown in The target hazard quotient (THQ) in beef was found to be significantly low in Cu, Zn, and Fe, while this was highest in Pb  was high in beef as the levels of Pb played a major cumulative risk in increasing the cancer threat in the population. Furthermore, the hazard index (HI) was high (HI\u2009>\u20091), thus showing that the cumulative effects of contaminants in the samples would be dangerous. Bearing in mind that high Pb levels have been emphasized by this study in both milk and beef, future studies in Uganda would offer a more descriptive picture since this study was only conducted in Bushenyi district. The low trace elements are of physiological benefit to human, and reasons as to why they are in low concentrations in these samples would help guide policy, for improved consumer protection.Observations made in this study show that chronic effects of the inorganic pollutants are a major public health threat, especially due to the strong cancer effects. Exposure to multiple contaminants results in additive and interactive effects; thus, the hazard index was used as an indicator of risk. Studies to include more areas in the region in order to determine the geographical extent of the threat at hand would have to be conducted; however, information in this study offers a probable cause to the increasing incidence of cancer within the Ugandan population, showing a need to revise current food safety policies and promote environmental protection."}
+{"text": "Profound and debilitating fatigue is the most common complaint reported among individuals with autoimmune disease, such as systemic lupus erythematosus, multiple sclerosis, type 1 diabetes, celiac disease, chronic fatigue syndrome, and rheumatoid arthritis. Fatigue is multi-faceted and broadly defined, which makes understanding the cause of its manifestations especially difficult in conditions with diverse pathology including autoimmune diseases. In general, fatigue is defined by debilitating periods of exhaustion that interfere with normal activities. The severity and duration of fatigue episodes vary, but fatigue can cause difficulty for even simple tasks like climbing stairs or crossing the room. The exact mechanisms of fatigue are not well-understood, perhaps due to its broad definition. Nevertheless, physiological processes known to play a role in fatigue include oxygen/nutrient supply, metabolism, mood, motivation, and sleepiness\u2014all which are affected by inflammation. Additionally, an important contributing element to fatigue is the central nervous system\u2014a region impacted either directly or indirectly in numerous autoimmune and related disorders. This review describes how inflammation and the central nervous system contribute to fatigue and suggests potential mechanisms involved in fatigue that are likely exhibited in autoimmune and related diseases. According to the National Institutes of Health, autoimmune diseases are estimated to afflict over 20 million individuals in the United States , 2. CurrFatigue is multifaceted and typically broadly defined making it difficult to decipher the causes in specific autoimmune diseases . FatigueCurrently, there is a lack of efficacious long-lasting treatments for individuals experiencing fatigue in autoimmune disease. This is due, in part, to the limitations in our understanding of the multiple mechanisms responsible for fatigue. Evidence suggests several physiological functions can contribute to fatigue including oxygen/nutrient supply , 23, metAutoimmune diseases are associated with enhanced pro-inflammatory signals in the periphery and CNS \u201337. The Cytokines are small proteins molecules involved in cell signaling allowing cells to communicate through autocrine, paracrine, or endocrine mechanisms . CytokinAutoimmune disease induces enhancement in cytokines such as IL-1\u03b2, TNF-\u03b1, IL-6, IL-12, IL-23, and IFN-\u03b3, especially by T helper cells and macrophages , 65. ConInflammation in the periphery can induce inflammation in the CNS and sickness behaviors , 69, whiIL-1\u03b2 binds to IL-1 receptor type I (IL-1R1) to induce inflammatory effects . IL-1\u03b2 cCognitive fatigue involves declines in alertness, orientation, and mental performance on cognitive tasks and is associated with feelings of exhaustion that follow sustained cognitive demands . IndividAutoimmune diseases including inflammatory bowel disease, multiple sclerosis, and rheumatoid arthritis have a high comorbidity with anxiety, depression, and pain , which cThe perception of effort and motivation can modify fatigue and are affected in autoimmune disease. Animal studies indicate that effort expenditure is influenced by inflammation \u2013106. In A primary mechanism that activates IL-1\u03b2 occurs through the activation of inflammasomes . InflammInflammasomes are involved in modulating behavior including sleep , cognitiIFN-\u03b3 is involved in innate and adaptive immunity. IFN-\u03b3 and its ligands (the IFN-\u03b3 receptor 1 and IFN-\u03b3 receptor 2) are found throughout tissues in the periphery and CNS, especially in macrophages, microglia, and lymphocytes . IFN-\u03b3 rMetabolism involves the conversion of fuel sources to energy-related molecules\u2014such as adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide phosphate (NADPH)\u2014enabling cellular processes; synthesis of components for proteins, lipids, nucleic acids, and carbohydrates; and the removal of ROS such as peroxides, superoxide, and hydroxyl radicals . MetabolATP is an energy molecule involved in the catabolism of macronutrients including carbohydrates, proteins, and lipids . ATP is ROS are involved in cell signaling, homeostasis, and normal physiological processes including inflammatory processes . EvidencEvidence indicates that NADPH oxidases are activators of NLRP3 inflammasomes . In partMitochondrial DNA dysregulation has also been observed in inflammatory conditions including autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, and granulomatosis with polyangiitis . MitochoThioredoxin (TRX) and its endogenous inhibitor thioredoxin-interactive protein (TXNIP) are involved in the activation of inflammasomes . TXNIP, The mechanistic target of rapamycin (mTORC) is a major sensor of metabolic stress and the pro-inflammatory response , 168. EvCyclooxygenase (COX) converts prostaglandins from arachidonic acid . COX-2 iA bidirectional relationship appears to exist between sleep and circadian disturbances with autoimmune disease \u2013175. ChrSleepiness, often referred to as drowsiness, can be loosely defined as the inability to remain awake or the enhanced occurrence or compulsion to sleep. Sleep disorders and disturbed sleep are associated with sleepiness . SleepinSleep disorders\u2014such as sleep apnea, insomnia, and REM sleep behavior disorder\u2014are associated with enhanced levels of inflammatory molecules \u2013210. SinRecently, the NLRP3 inflammasome was found to be critical to sleep and electroencephalogram delta power fluctuations throughout the day as well as responses to sleep loss and inflammatory stimuli in mice . Additio+/IFN-\u03b3+ T cells with the loss of myeloid BMAL1 at midday instead of midnight  and CLOCK (CLK) form heterodimers in the cytoplasm that regulate gene expression oscillator properties of cells that regulate both the persistence and duration of circadian rhythms . BMAL1 amidnight . These dMelatonin is secreted by the pineal gland, regulates circadian rhythms, and has anti-inflammatory and anti-oxidant properties . MelatonThe hypothalamic-pituitary-adrenal (HPA) axis involves interactions between the hypothalamus and pituitary glands and is involved in hormonal responses to stress that modulate fatigue , 227. Inin vitro suggesting relationships exist between the activation of purinergic and adrenergic systems  . CRH indA receptor (The weak androgen dehydroepiandrosterone (DHEA) produced in the adrenal cortex. The HPA-axis, in part, controls DHEA synthesis . The grereceptor . DHEA isreceptor , 242. DHSeveral brain areas are associated with modulating fatigue  243), a, a243), A receptors that are ligand-gated ion channels and GABAB, which are metabotropic receptors that are G protein-coupled receptors that open and close ion channels. GABA functions to help neurons recover after transmission. It has been shown to reduce anxiety and stress and is involved in sleep and sleepiness  . HistamiGlutamate is a major excitatory neurotransmitter found throughout the CNS that is produced by metabolism . GlutamaAcetylcholine is an excitatory neurotransmitter that is synthesized by choline acetyltransferase from choline and acetyl-CoA . AcetylcArousal and wakefulness involve an ascending pathway that begins in brainstem monoaminergic and cholinergic neurons located between the pons and the midbrain in the mesopontine junction . FatigueOrexin  is a neuropeptide that binds to orexin 1 and orexin 2 receptors . These nTwo mechanisms whereby peripheral inflammation can enhance CNS inflammation are through leaky areas in the blood-brain-barrier and the vagus nerve . The vagThe vagal nerve afferents project to the dorsal vagal complex, which consists of the NTS, the dorsal motor nucleus (DMN) of the vagus, and the area postrema, located in the medulla area of the brainstem. However, the NTS is the primary area of vagal afferent stimulation. Neurons in the brainstem project toward many areas of the brain that can modulate fatigue including the amygdala, cortex, central nucleus of the amygdala, nucleus accumbens, paraventricular nucleus, and lateral hypothalamic areas of the hypothalamus, cerebellum, and other areas of the brainstem , 315. ThStudies in rats and mice have demonstrated that the vagal afferent stimulation by IL-1\u03b2, TNF-\u03b1, or LPS delivered IP can enhance inflammatory IL-1\u03b2 or TNF-\u03b1 gene expression in the brain , 318. CoVagotomy does not inhibit central inflammation induced by peripheral inflammation by large concentrations of inflammatory stimuli , suggestThe DMN and NTS are major sources of efferent motor vagal input. Longer preganglionic cholinergic neurons communicate with postganglionic neurons in closer proximity and within tissues of the viscera to induce anti-inflammatory signals . AcetylcVagal efferent nerves project to the reticuloendothelial system, other peripheral organs, and the brain-derived motor output . AcetylcInterestingly, evidence suggests relationships between peripheral muscle weakness and pain, inflammatory pathways, neuroinflammation, and fatigue in certain autoimmune disorders. A recent study found that serum TNF-related apoptosis-inducing ligand, which is a TNF-superfamily member, is enhanced in the serum and expressed in infiltrating inflammatory cells in patients with polymyostitis and dermatomyostitis \u2014conditio2), signaling molecules, and provides adequate supplies of energy reserves , Wegener's granulomatosis, Churg-Strauss syndrome, Good pasture's syndrome, and ankylosing spondylitis are known to induce inflammation in the lung and alter lung functions . MacrophHyperventilation can induce fatigue and is implicated in perpetuating chronic fatigue syndrome/myalgic encephalomyelitis . A condiA complex and not well-understood relationship exists between the brain vasculature, cerebrospinal fluid, and the perivascular spaces that act as lymphatics in the brain . In 1968There are several areas of research and logistics that need to be established to understand the exact mechanisms of fatigue and sleep in autoimmune diseases. First, a detailed description of particular types of fatigue needs to be established in the clinic. This can be achieved, in part, with more precise medical coding. Second, there needs to be standardized questionnaires and diagnostic tests that can more precisely indicate the determents observed from fatigue. This will provide insight into the types of fatigue that are observed with the pathogenesis of the autoimmune disorders. Understanding the neurocircuitry of fatigue and its relationship between inflammation and autoimmune diseases is also needed. This area can aid in understanding the manifestation of types and severity of fatigue found in autoimmune patients. Since inflammation and metabolism are implicated in fatigue and impairments are found in these in autoimmune disorders, a detailed understating of the mechanisms of these systems, unique cells, and brain areas are lacking in autoimmune research. More information on interactions between circadian timing and sleep/wake state or sleep loss, alterations in neuronal activity, and normal daily functioning that affect fatigue is required. And there needs to be a better understanding of the relationship between the vagal afferents in modulating brain inflammation to induce fatigue is needed. More research is also necessary for understanding the relationship between vasohemodynamics and fatigue in autoimmune disease. Additionally, several autoimmune diseases are associated with disproportionately greater incidence and disease severity in particular genders and little information is known regarding the relationship of fatigue with these gender differences . The relAn autoimmune disease involves multiple interactions between genetics and environmental factors. Most autoimmune-related disorders are more prevalent in monozygotic twins vs. dizygotic twins or siblings, indicating the involvement of genetics in disease development. Furthermore, genome wide association studies (GWAS) have found several genetic loci and small nucleotide polymorphisms that are associated with specific autoimmune disease prevalence. Interestingly, proteins encoded by genes that are involved in inflammatory mechanisms including NF-\u03baB, apoptosis, Toll-like receptor, and immune complexes are described in autoimmune and related disorders. Nevertheless, a number of autoimmune diseases exhibit similar genetic modifications, which likely contributes to the higher incidence of multiple autoimmune diseases found in individuals with autoimmune disorders and potentially similar disease characteristics including fatigue. Indeed, larger data sets  are finding new associations of inflammatory mechanism I with impairments in respiratory function and sleep . SeveralIn summary, fatigue is a major early finding in individuals with autoimmune diseases, and inflammation is a contributing factor relating to this impairment, one that affects the ability of people to perform daily activities, work, and thus their overall well-being. Recent research reveals a relationship between types of fatigue and certain brain areas, cell types, and phenotypes that mediate the symptoms observed. In addition, inflammatory molecules that are enhanced in the periphery with specific types of autoimmune disease can alter brain inflammation and neurocircuitry affecting fatigue. Consequently, immunomodulatory agents and drugs targeting inflammatory pathways could serve to treat fatigue occurring in autoimmune and related diseases. Understanding the mechanisms behind fatigue will not only aid individuals with autoimmune diseases but could also benefit transplant recipients, cancer patients, and infectious disease patients who experience debilitating fatigue.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Acripeza reticulata to test the efficacy of a putative deimatic display in the wild. Mountain katydids have a complex defence strategy; they are camouflaged at rest, but reveal a striking red-, blue-, and black-banded abdomen when attacked. We presented live katydids to sympatric (experienced) and allopatric (naive) natural predators, the Australian magpie Cracticus tibicen, and observed bird reactions and katydid behaviors and survival during repeated interactions. The efficacy of the katydids\u2019 defence differed with predator experience. Their survival was greatest when faced with na\u00efve predators, which provided clear evidence of the protective value of the display. In contrast, katydid survival was consistently less likely when facing experienced predators. Our results suggest that sympatric predators have learned to attack and consume mountain katydids despite their complex defense, and that their post-attack display can be an effective deterrent, particularly against na\u00efve predators. These results suggest that deimatism does not require predator learning to afford protection, but that a predator can learn to expect the display and subsequently avoid it or ignore it. That sympatric predators learn to ignore the defense is a possible explanation for the mountain katydid\u2019s counter-intuitive behavior of revealing warning colors only after tactile stimuli from predator attack.Predation has driven the evolution of diverse adaptations for defence among prey, and one striking example is the deimatic display. While such displays can resemble, or indeed co-occur with, aposematic \u2018warning\u2019 signals, theory suggests deimatic displays may function independently of predator learning. The survival value of deimatic displays against wild predators has not been tested before. Here we used the mountain katydid  Particular progress has arisen from work on aposematic systems, in which prey advertise their unpalatability to predators via static  colour patterns9. Defensive displays, however, can include multiple elements such as a sudden transition between camouflage and aposematism, or the dynamic presentation of an otherwise concealed defence16. It has been suggested that a sudden transition could constitute a deimatic component of such defensive displays, but empirical data required to test this idea are scarce23. There are likely profound differences between the aposematic and the deimatic components, with respect to the mechanisms by which they deter predators (see below) and these differences have ecological and evolutionary implications, for which there is a paucity of data23.The study of visually conspicuous defensive signals has driven the development of fundamental evolutionary theory in predator-prey interactions24. In the case of aposematism, na\u00efve predators must initially learn to associate prey signals with defenses - unless they exploit pre-existing biases - which reduces the likelihood of future encounters escalating to an attack26. The protective value of an aposematic signal thus tends to increase as a function of predator experience. It has been predicted that due to its sudden transition, deimatism does not require predators to accumulate experience, and therefore that its protective value is greater against inexperienced predators compared to that of aposematism17. If deimatic prey do not deploy a protective secondary defence such as weaponry or chemical defenses, the efficacy of their display could decrease as a function of predator experience, if predators learn to suppress their aversive response27. Alternatively, this effect may be ameliorated by the presence of further co-occurring defences, such as an aposematic element combined with a deimatic element.The adaptive value of defensive signals lies in their ability to interrupt the predation sequenceHere we tested the role of predator experience in shaping the protective value of a defensive, putatively deimatic, display in the wild. We examined the responses of a known avian predator to an insect in assays designed to quantify both the absolute and relative protective value of the display across natural variation in predator experience. If the display exploits a reflexive response in the predator, which does not require learned aversion, we predicted that upon first presentation na\u00efve predators should be less likely to attack, kill, and eat the prey than experienced predators. If, in contrast, the display is solely reliant on the formation of learning rules in a manner akin to aposematism, then its protective value should be greatest against experienced predators. Further, we predicted that experienced predators would respond consistently between their first and second encounter with katydids, while na\u00efve predators would attack the unknown prey on their first interaction and, once startled, abandon the hunt. On their second encounter, we expected previously na\u00efve predators to either: (a) avoid the insect, if they learned to associate the display (and/or co-occurring defence) with unprofitability; or (b) attack the insect, if they learn to expect the display and the insect is sufficiently profitable prey.Acripeza reticulata, Gu\u00e9rin-M\u00e9neville 1832) is a large, putatively chemically defended orthopteran native to the montane regions of south-eastern Australia29. Mountain katydids have tough cover wings (tegmina) and are cryptic at rest, but reveal a striking red-, blue-, and black-banded abdominal pattern when attacked  but the effect of those alkaloids on predators is currently unknown. The Australian magpie, Cracticus tibicen, is a known primary threat to mountain katydids in the wild, and we therefore selected them as our focal predator31. Australian magpies are easily habituated to humans33, common both within and outside of the mountain katydid\u2019s range29, and have a generalist omnivorous diet that includes ground dwelling insects32. Magpies hold small (~2\u2009km2), distinct, stable, year-round territories that rarely overlap with conspecifics32. Their plumage is highly variable and sexually dimorphic, allowing the reliable identification of individuals in a small family group. By never visiting a territory more than once we further ensured independence of trials. We conducted trials with Australian magpies sympatric to mountain katydids in human-inhabited areas of Kosciuszko National Park, NSW , and with allopatric magpies throughout the suburbs of Sydney . Though the genetic structure of groups and relatedness of individual magpies within each location is unknown, our current understanding of their ecology (as above) strongly suggests that we sampled from numerous populations within these broad zones of \u2018sympatry\u2019 and \u2018allopatry\u2019. As detailed below, we also include location effects in our statistical analyses, thus controlling for any possible cultural transmission and social learning among family groups. We targeted magpies in Kosciuszko National Park that held territories within or close to ski resorts (~500 year-round residents), camping grounds, and caravan parks, both to ensure that they were habituated to humans and that they had access to ample food. The magpie populations within both Kosciuszko and Sydney often displayed \u2018urbanised behaviours\u2019 such as foraging in bins, picnic tables, and taking food from people. Through this approach, the birds from Sydney and the birds from Kosciuszko were comparable, despite being wild, free ranging predators. In many of the sympatric magpie territories, mountain katydids have been seen by us , could be heard calling, and/or their Senecio spp. host plants were visible when experiments were taking place. Thus, we expect populations of sympatric magpies had experience with mountain katydids. The sympatric and allopatric ranges are separated by approximately 400\u2009km. We ran the experiment concurrently across both sympatric and allopatric magpies through March and April, 2016. We habituated magpies using small pieces of cheese as rewards before the commencement of trials to enable us to approach close enough to gently present stimuli.In addition to their visual display, mountain katydids regurgitate their crop contents, and exude a bitter tasting liquid from their abdomen. Both are likely rich in alkaloids derived from their Senecio gunnii and S. linarifolius in Kosciuszko National Park in March and April 2016. Mountain katydids are sexually dimorphic and females were chosen because, unlike males, they cannot fly and have a very large striking blue and red display , thereby ensuring the maintenance of their putative chemical defence and similar levels across all katydids used in the experiment. No katydid was held for longer than seven days before being used in an experimental trial.We collected 264 female mountain katydids from patches of ay Video\u00a0. We housKosciuscola tristis (~0.5\u2009g), a common insect in sympatric locations; or house crickets, Acheta domesticus (~0.5\u2009g), a common pest insect in allopatric locations); (3) an inedible stimulus, to control for the conditioning of wild birds to humans as sources of food . Unpalatability is always relative, so we chose an inedible control and a palatable control with the expectation that the katydid would be more acceptable than the inedible control and less acceptable than the palatable control. The inedible stimulus also provided a negative control that was undeniably less profitable than a katydid. Among sympatric birds, we ran trials in the fixed order outlined above  to minimise bias toward individual predators. For the allopatric birds we ran the trials in a slightly different order , to ensure that the katydids we relocated to Sydney were not held for longer than seven days.We assigned habituated predators to one of three treatments, defined by the focal stimulus being presented. For a given trial, we presented each individual predator with a stimulus from their allocated treatment twice, with an interval of two minutes between presentations. We minimized the potential for social learning by luring away all individuals in the immediate vicinity of the test subject (ca. 10\u2009m) using small pieces of cheese as bait before treatment presentation. The test stimulus was one of: (1) a mountain katydid (2.38\u2009\u00b1\u20090.52\u2009g); (2) a palatable orthopteran , so we excluded these trials from our analyses. We also excluded trials in which magpies failed to notice the stimulus at the beginning of either presentation (ca. 4%), and a single trial in which a na\u00efve bird avoided the katydid before seeing their display, with a final total of 163 complete trials.We scored magpie behavior at the time of the presentation , and afterwards from video footage collected during the trials Video\u00a0. We eval34. We modelled the responses of magpies coded on a scale of one to five (detailed above) as an ordinal response variable, and included treatment , presentation number (one/two) and predator experience  as main effects, with location and magpie-ID as random effects. We used log-likelihood ratio \u03c72 to test the significance of the overall model and individual predictors. All analyses were run in R35, primarily using the clmm function in the package ordinal (v2018.4-1937). All p-values quoted are two-tailed.We used mixed-model ordinal logistic regression to test the effects of treatments, experience, and presentation number on the probabilities of the behavioral escalation of magpies during predationAll experiments were conducted in accordance with relevant guidelines and regulations under ethics permit number: AE14/35 from the University of Wollongong.2\u2009=\u200915.59, df\u2009=\u20096, P\u2009=\u20090.016; Fig.\u00a0In total, we presented stimuli to 163 different, free-ranging, wild Australian magpies, two of the same stimuli per bird. Of those sympatric to mountain katydids, we presented 29 magpies with plasticine balls, 24 magpies with local grasshoppers and 29 magpies with mountain katydids. To allopatric magpies we presented 21 magpies with the plasticine ball, 23 magpies with house crickets and 37 magpies with mountain katydids. Any differences in the sample sizes for each treatment were due to ensuring that we used as many katydids as quickly as possible so we did not hold them for more than 7 days. The responses of magpies differed between experimental treatments, individual stimulus presentations, and the extent of prior experience with katydids as estimated by sympatry or allopatry  the toxin load is worth the trade-off40, or (c) because sympatric predators learned to ignore the katydids\u2019 defences over many encounters, suggesting that they are not as well-defended as they seem27.In addition to demonstrating the effectiveness of the katydid\u2019s display, our data support our prediction that experienced predators respond consistently toward katydids between the first and second presentations. Unexpectedly, however, rather than avoiding these seemingly well-defended insects, experienced (sympatric) predators were more likely to attack, kill and consume katydids than na\u00efve predators. This indicates that katydids are in fact profitable prey for experienced predators, or at least relatively so, because (a) they can physiologically tolerate or behaviorally remove their chemical defense25. The katydid\u2019s effective defense against na\u00efve predators is unlikely to be explained by neophobia, because the majority of magpies approached and attacked the katydids while the katydids were in their camouflage phase. Upon second presentation, however, allopatric predators avoided katydids, seeming to have learned to avoid them from their first experience.On first presentation, katydid defenses were far more effective against na\u00efve, allopatric predators, than experienced, sympatric predators. This result is consistent with the hypothesis that deimatic displays do not require predator learning, in contrast to what is generally expected for aposematism42. These explanations are unlikely, however, as ample alternative food sources were available during the time we conducted the experiment, late summer, and within the wide range of both sympatric and allopatric magpies43. There is a considerable diversity of aposematic invertebrates in the range of our allopatric magpies, which would offer opportunity for experience with defended prey, for example the transverse ladybird, Coccinella transversalis. However, the distinctiveness of the mountain katydids\u2019 appearance44 minimises any potential for Batesian mimicry to provide an explanation for our results. We do acknowledge, however, that while individual magpies were drawn from a large geographic area relative to their known territory size  it would be valuable to extend this experiment using entirely independent predators across a more closely controlled range of experience, to further examine the above explanations.Sympatric and allopatric predators reacted to the presentation of mountain katydids differently. Sympatric predators were more likely to attack, kill and eat mountain katydids compared to allopatric predators. We see two potential, non-mutually exclusive explanations for this pattern: (1) the behaviour of the predators reflects regional variation, (2) predator experience influences the efficacy of the display. Differences in sympatric and allopatric magpie behavior may reflect regional variation in predator ecology, such as local adaptation, poorer condition or less discerning sympatric predators, or heightened neophobia and/or wariness among allopatric predators24. Below, we discuss both possibilities, and a third, that the defense has both deimatic and aposematic components, making it a multi-component display.Our results are consistent with two mechanisms by which the mountain katydid\u2019s display provides protection; deimatism and aposematism17. Deimatism is not the only mechanism by which prey might be protected from na\u00efve predators, since initial avoidance can also be facilitated by forms of neophobia and dietary conservatism1. However, under these mechanisms, one may not expect the predator to approach and attack novel prey as they did in our study, reducing the likelihood of this hypothesis in our case. Nevertheless, protection by their display against na\u00efve predators is not predicted under aposematism, which is usually described as a learned signal1.Upon first presentation of a katydid, na\u00efve allopatric predators were less likely to attack, kill and eat katydids than experienced sympatric predators. Recent suggestions predict that deimatism should be effective against na\u00efve predators because it is thought to exploit \u2018hard-wired\u2019 reflexive responses that do not require learninget al.27 showed declining protective value of auditory startle with repeated interactions between red-winged blackbirds (Agelaius phoeniceus) preying on walnut sphinx caterpillars (Amorpha juglandis)27 suggesting that predators learn to ignore the startle, but whether their result is generalisable across modes and taxa remains to be seen.Allopatric predators reduced their attack rate markedly in the second encounter compared to the first. This is expected under aposematism, where predators are less likely to attack after an adverse experience, however, this is not necessarily inconsistent with deimatism. In principle being \u2018startled\u2019 might be aversive enough for predators to avoid future interactions, especially given the relatively short temporal window between presentations in our experiment (2\u2009min), which could have meant that a startle effect still lingered in the predator\u2019s mind upon the second encounter. Whether this occurs in nature is currently unclear because there is little evidence as to whether deimatism enhances or inhibits learning. If we assume startle can be learned, it is unclear whether predators learn to pay attention to it or learn to ignore it. Dookie 16, reducing the likelihood of a subsequent approach.If the katydid\u2019s defence is a combination of deimatism and aposematism, it is possible that during their first encounter in which the magpies attacked the katydids and were startled, the magpies tasted the katydids, saw their display and learned to associate those experiences with the rest (putative camouflage) phase, thus reinforcing aposematism. Therefore, the allopatric predator response can be explained if we consider that the katydid\u2019s defence has multiple components: in the first instance the display deters predators based on reflexive recoil, but it also reinforces the association between bad taste and colorful signal17. There are several non-exclusive facets to this explanation. One is that sympatric, experienced predators may have learned to suppress their reflexive aversion to the deimatic component of the katydid\u2019s display, at least in part, by expecting it. The circumstances or number of trials this might take is currently unknown. Our study is limited to two interactions, further experiments that aim to tease apart aposematism and deimatism may need to test predator responses in a series of interactions. This is consistent with work on vertebrate predators showing that, while highly sensitive to movement in general, they may rapidly learn to disregard non-salient motion cues following repeated exposure47. We further suggest that the katydid\u2019s display reveals an aposematic signal, but that sympatric, experienced predators have behavioural or physiological strategies to resist chemical defences or they may be able to consume chemically defended prey by making strategic decisions about when to incur the assumed cost of consuming them48. Two observations of Australian magpies living sympatrically with mountain katydids suggest that at least some individuals wash31 and wipe (Umbers and De Bona pers. obs.) them before consumption which may imply that sequestered compounds are stored in secretions rather than body tissue. Another possibility is that the mountain katydids\u2019 chemical-defence is less distasteful or toxic than has been suggested making them a relatively undefended deimatic displayer28. Their diet of alkaloid-rich Senecio, and the strong aversive response of na\u00efve predators upon secondary presentation . Thus, despite any chemical defence, the best strategy for many prey is not to be overtly conspicuous50 or perform their display too often, if its efficacy is partly based on predator inexperience.Many features of deimatic displays seem counter-intuitive. For example some species, such as the mountain katydid, wait until they have been physically attacked before performing their display52, unravelling the mechanisms by which they confer protection, and how they evolve, remains a challenge17. Teasing apart the various components of defensive displays and understanding how they work in concert is a major challenge for this field. Among predators, innate aversive responses to threatening stimuli are ubiquitous, and arise early in development57. Such perceptual biases are well known to serve as the substrate for signal evolution, though this has chiefly been explored in the context of sexual communication59. Male field crickets (Eneopterinae: Lecithin), for example, seemingly exploit the startle-responses of females by generating high frequency calls that are typically associated with local predators60. These calls goad females into producing a vibrational response, which males can then use to locate them. Similarly, our results support the notion that the existence of reflexive biases in predators can generate \u201cevolutionary opportunities\u201d for exploitation in defensive strategies (Fig.\u00a023. This stands in contrast to aposematic systems which, owing to their reliance on general learning rules in predators, are thought to require the evolution of prey defences among cryptic ancestors prior to conspicuous signals61. However, this does not mean that deimatism and aposematism are mutually exclusive, we expect that in many species they could work in concert. Ultimately, our results offer some early empirical support for the protective value of a complex defensive display and provide a strong field-based system with which to understand the mechanistic side of these charismatic defences.Despite a growing appreciation of the importance of defensive displaysies Fig.\u00a0. These oThis experiment was conducted under ethics permit AE14/35.Supplementary Dataset 1Supplementary Video S1"}
+{"text": "Anxiety is among the most prevalent and disabling mental health problems in older adults. Few older adults with mild to moderately severe anxiety symptoms receive adequate interventions, putting them at risk for developing anxiety disorders, depression, and various somatic problems. Effective, low-threshold interventions should be developed. Blended care, in which a web-based intervention is combined with a limited amount of face-to-face contacts with a mental healthcare counselor at the general practice, is a promising option. The online self-help intervention \u201cLiving to the Full\u201d\u2014an Acceptance and Commitment Therapy (ACT) intervention\u2014has been proven to reduce depression and anxiety in several patient groups, but has not yet been investigated in older adults. The aim of this study is to evaluate the (cost-)effectiveness of a blended form of \u201cLiving to the Full\u201d in reducing anxiety symptoms in adults aged 55 to 75 years. Furthermore, moderators and mediators of the treatment effect are investigated.The (cost-)effectiveness of the ACT intervention will be investigated in a cluster single-blind randomized controlled trial (RCT). The blended intervention will be compared to treatment-as-usual. Thirty-six mental health counselors working at general practices in the Netherlands will be randomized to deliver blended care or treatment as usual. A total of 240 participants (aged 55\u201375\u00a0years) with mild to moderately severe anxiety complaints  will be recruited. There are four measurements consisting of online questionnaires (primary outcome: GAD-7) and a telephone interview: before the start of the intervention; directly following the intervention (14\u00a0weeks after baseline); and six and twelve months after baseline. Possible mediator variables will be assessed multiple times basis during the intervention.This RCT will evaluate the effectiveness of a blended ACT intervention for older adults with anxiety symptoms. If the intervention is shown to be effective, it will be implemented, thereby improving the accessibility and quality of preventive interventions for older adults with anxiety problems.NTR6270. Registered on 21 March 2017.Netherlands Trial Register, The online version of this article (10.1186/s13063-018-2731-3) contains supplementary material, which is available to authorized users. Anxiety problems form one of the most ubiquitous and disabling mental health conditions in older adults. Prevalence estimates of anxiety disorders in this age group are in the range of 1.2\u201314% . This isThe fact that only a small proportion of anxious older adults receive adequate treatment is alarming, since even on a subclinical level, anxiety in older adults is associated with diminished quality of life and wellbeing , 8, deprIn younger adult populations, Internet-based therapy\u2014predominantly cognitive behavioral interventions\u2014have repeatedly been shown to form a (cost-)effective treatment option for both anxiety disorders and subclinical anxiety \u201325. EvidTo date, only one study has examined an online intervention for older adults with anxiety symptoms. This randomized controlled trial (RCT) found that Internet-delivered cognitive behavioral therapy (CBT) was an efficacious and cost-effective treatment for older adults with subclinical anxiety . The vasOne treatment approach that might form a valuable alternative for CBT in older adults is Acceptance and Commitment Therapy (ACT), a so-called third-wave CBT , 33\u201335. Considering the need for evidence-based interventions for older adults with subclinical anxiety and the plausible suitability of ACT for this group, the proposed study aims to evaluate the (cost-)effectiveness of an Internet-based ACT intervention for older adults with anxiety complaints in general practice. To promote compliance and reduce dropout, this intervention is combined with a limited amount of face-to-face contacts with the mental health counselor at the general practice. This intervention will be compared to optimized treatment-as-usual. In addition to the analysis of the (cost-) effectiveness, moderators and mediators of treatment effects will also be investigated in the study. The current paper describes the research protocol for this study.The proposed study is a pragmatic cluster single-blind RCT. In a clustered trial, the unit of randomization is not the individual participant, but a group of participants . In this study, randomization takes place on the level of the mental health counselors at general practices, which creates clusters of participants that receive treatment from the same counselor. Cluster randomization is used in this study to reduce experimental contamination  and to mR software. Participants, mental health counselors, and the main researcher  cannot be blind for allocation to conditions. However, participants are not informed on whether the treatment they receive is the experimental or active control intervention. They are told that the study aims to compare two treatment options for people with mild to moderate anxiety complaints. This is considered an ethically sound approach, since treatment-as-usual is delivered as optimized care as usual.The randomization table  will be created by an independent researcher using Each participant will complete four main measurements during the study: before the start of the intervention (and before participants are informed about which intervention they will receive) (T0); directly following the intervention ; and six (T2) and twelve months (T3) after baseline. All measurements consist mainly of online self-report questionnaires. T0, T1, and T3 also include a telephone interview conducted by a research assistant. The research assistant that conducts the telephone interviews will be unaware of treatment allocation of the participants. If for any reason this blinding is broken, another research assistant (that is blind to treatment condition of the participant) will take over the telephonic assessment.Figure A total of 240 participants will be included. In order for a person to be eligible to participate in the study, the following inclusion criteria have to be met: aged 55\u201375\u00a0years; presence of mild to moderate anxiety symptoms; access to Internet; mastery of the Dutch language; and the possibility and motivation to spend up to 30\u00a0min per day on the intervention. Potential individuals that meet any of the following exclusion criteria will be excluded from participation: Alzheimer\u2019s disease or other severe cognitive impairments; unstable severe medical condition(s); severe anxiety or few anxiety symptoms; severe depressive symptomatology; having received psychological or psychopharmacological treatment (with the exception of stable use of benzodiazepines or SSRIs) within the last three months; severe role impairment in at least two areas of role functioning; high suicide risk; substance use disorder or a lifetime diagnosis of bipolar disorder or schizophrenia. The main inclusion criterion\u2014severity of anxiety symptoms\u2014is measured with the GAD-7. This questionnaire uses well-established cut-off points for mild, moderate, and severe anxiety symptoms . These cCluster randomization will be applied at the level of the participating mental health counselors. Assuming a mean cluster size of five patients per counselor, an intraclass correlation coefficient of 0.01  and a coGeneral practices will be recruited throughout the Netherlands, with a focus on the Leiden and the Hague area. Both general practitioners and the mental health counselors working at the general practices will receive a printed invitation to participate in the study, after which they will be contacted by email and phone to recommend participation. When a practice  agrees to participate, the mental health counselor will be randomized. A total of 36 mental health counselors will be included. Randomization is performed by an independent researcher, who is informed by the main researcher when a group of four mental health counselors have been enrolled. The mental health counselors are informed about their group allocation by the main researcher and are invited to attend a training in the treatment method they will apply during the study.A data manager will visit the participating general practices to assist the general practitioner in the selection of patients aged 55\u201375 years that are eligible to receive an invitation letter for the study. Patients whose medical records mention a lifetime diagnosis of bipolar disorder or schizophrenia, a severe unstable medical condition, Alzheimer\u2019s disease, or other severe cognitive impairments will not receive an invitation. All other patients aged 55\u201375\u00a0years do receive an invitation letter. The invitation letter from the GP to the patients contains information about anxiety complaints and explains the aim, design, and procedure of the study. Furthermore, the letter refers to the study website. On this website, potential participants can read more extensive information about the study and indicate their interest to participate. After doing so, they will be asked to fill in the GAD-7 and PHQ-9 and to indicate whether they have received psychological treatment for emotional problems during the last three months, have Internet access, have sufficient time for treatment, are able to communicate in Dutch, and are aged 55\u201375\u00a0years. If based on these answers patients are still eligible to participate, they are asked if they are willing to be interviewed by phone to determine whether they fulfill all inclusion criteria. They are asked to fill in their telephone number and to indicate on which day they prefer to be called. Within ten days they will be phoned by a research assistant, who will administer the SDS and M.I.N.I.-Plus. Furthermore, they will be asked about their medication use. Patients will also be given the opportunity to ask any remaining questions about the aim of the study and the study procedures. The interviewer will discuss his/her diagnostic findings from the M.I.N.I-Plus with a senior clinical psychologist; subsequently the interviewee is informed by mail about inclusion or exclusion. When patients are eligible, they will receive a link to an online informed consent form and the baseline questionnaires (T0). Ineligible patients will be referred to their general practitioner in case they want help for their complaints. Once eligible patients have filled in the informed consent form and the baseline questionnaires, the mental health counselor at their general practice will be informed about study participation and they will be invited for a first appointment. In addition to the mass mailing, general practitioners and mental health counselors will recommend participation to patients aged 55\u201375 years that attend the practice with anxiety complaints during the inclusion period. They will inform these patients about the study and refer them to the study website if they want to participate. These patients then follow the screening and inclusion procedure as described above.During the intervention, participants will fill in a short questionnaire assessing potential mediator variables several times. At these moments they will also complete the GAD-2  and PHQ-Both interventions are delivered in a timespan of maximum 12 weeks, after which participants receive an e-mail with a link to the online self-report questionnaires of the post-treatment assessment (T1). T1 also includes a clinical interview by telephone conducted by a research assistant. The follow-up assessments take place six (T2) and twelve months (T3) after baseline. Both these assessments consist of self-report questionnaires and T3 also includes a clinical telephone interview. Figure Although available research shows that the effect of eHealth interventions does not depend on age and that older adults are even more adherent than younger adults , severalThe online intervention \u201cLiving to the Full\u201d is an adaptation of the similarly titled ACT-based self-help book  , cognitive or behavioral aspects typical of most anxiety complaints , or consequences of anxiety . All interventions consist of psycho-educative texts and exercises based on CBT. During the first session, the mental health counselor and participant make an overview of the psychological complaints of the participant. Based on this inventory, one or more of the small interventions are selected to form the basis of the next three sessions. In order to prevent treatment diffusion, the delivery of interventions resembling the web-based interventions  are not allowed. Mental health counselors will complete a short online questionnaire that measures treatment protocol adherence after every session. Mental health counselors in this condition will follow a training from an experienced clinical psychologist, to practice with the protocol. They will also be supervised during the study.Table\u00a0The primary outcome\u2014severity of anxiety symptoms\u2014will be assessed with the GAD-7 . This quFurthermore, during treatment, participants will fill in the GAD-2  several Depression symptoms will be measured with the PHQ-9 . This quPresence of current and/or lifetime diagnosis of anxiety disorder , obsessive-compulsive disorder, depressive disorder, post-traumatic stress disorder, and illness anxiety disorder will be determined by the M.I.N.I.-Plus . CurrentThe SDS  was deveThe subscales Rumination, Catastrophizing, Positive reappraisal, and Blaming yourself of the CERQ , 67 willThe Acceptance and Action Questionnaire (AAQ-II) , 72 is aThe Mental Health Continuum-Short Form (MHC-SF)  is a 14-The Five Facet Mindfulness Questionnaire- Short Form (FFMQ-SF)  will be The EuroQol-5 Dimensions-5 Levels questionnaire (EQ-5D-5\u00a0L) measures general quality of life using five dimensions  . Each diFor calculating the total direct medical costs, the Trimbos/iMTA questionnaire for Costs associated with Psychiatric Illness (TiC-P)  will be The Client Satisfaction Questionnaire-8 (CSQ-8)  is an eiStarting from existing instruments, Bovier, Chamot, and Perneger used factor analyses to develop four brief scales for the assessment of self-esteem, affective social and confident/problem-solving social support . All 12 A self-developed questionnaire will be used to measure negative life events and associated distress. Participants are asked if they have experienced negative life events in the past six months and/or earlier in their life. If they indicate that they have experienced such (an) event(s), they are asked to rate them on an 11-point scale  to what extent these experiences currently still evoke strong negative feelings.Co-morbid physical problems will be measured with a self-developed questionnaire listing\u00a025 (chronic) conditions. This list is based on information from Statistics Netherlands. Participants will also be asked to rate to what extent their somatic problem(s) interferes with their current daily functioning on an 11-point scale ranging from 0  to 10 (extremely).Using a self-developed questionnaire, the following socio-demographic information and additional information will be collected: age; gender; nationality; marital status; living conditions; education; work status; computer usage; and Internet usage.Participants\u2019 expectations of the treatment will be measured with a questionnaire derived from the Treatment Credibility Questionnaire (TCQ) from Borkovec and Nau . In thisA self-developed questionnaire measures the use of the following emotion regulation strategies: distraction; reappraisal; acceptance; rumination/worry; and suppression. Adaptive strategies  are consistently more strongly related with reduced negative affect and maladaptive strategies (rumination/worry and suppression) with enhanced negative affect in laboratory studies  and psycSince both interventions stimulate participants to limit their avoidance behavior, this factor will be included in the mediation analyses. Behavioral avoidance is measured with one item (i.e. \u201cIn the past week my anxiety caused me to avoid situations and/or activities\u201d), that participants rate on a 6-point-scale ranging from 0 (never) to 6 .Treatment expectancy is measured with one item: \u201cHow confident are you that the course will be helpful in reducing your anxiety complaints?\u201d Participants rate this question on a 7-point scale ranging from 1  to 7 (very confident). Several studies have shown the impact of client expectancies on treatment effect. Evidence suggests that eliciting hope and positive expectations about the effect of the treatment is a crucial factor in many psychotherapies \u201396.).Preliminary results suggest that the outcome of psychological interventions may be mediated by patient\u2019s self-efficacy with regard to the treatment (i.e. one\u2019s judgment of the capability to successfully participate in and complete the treatment) , 98. SelThe Session Rating Scale (SRS)  will be All analyses will use intention-to-treat principles and a two-tailed alpha of 0.05 for significance testing. Cluster randomization at the level of mental health counselors  results in a lack of independence for the outcomes of patients receiving treatment from the same counselor. If clustering and dependence of outcome are ignored, this could lead to underestimation of standard errors and regression coefficients . TherefoTo examine moderators of the treatment outcome, longitudinal multi-level regression analysis will be performed. Potential moderators will be entered individually in the model. Interaction effects will be investigated . Bivariate latent difference score models will be used for the mediation analysis. This type of analysis is recommended for repeated measurements and multiple mediators, because they allow for a more dynamic approach to mediation, by assessing changes in multiple variables and their interrelations over time \u2013105. CosThe proposed study will evaluate the (cost-)effectiveness of the Internet-based ACT intervention \u2018Living to the Full\u2019 for older adults with anxiety complaints in a RCT. This study has several strengths. First, the trial is unique in this field of research. It differs from the majority of studies in older anxious adults with regard to therapy approach (ACT instead of CBT), the delivery of the investigated intervention (Internet-based instead of face-to-face) and the study population (people with anxiety complaints instead of anxiety disorders). The moderation and mediation analyses form a second strength. These analyses will provide insight into respectively the effect of personal characteristics on treatment outcome and the processes through which the intervention achieves its effects. Including such analyses broadens the scope of the study. The trial is not merely focused on answering the question if \u201cLiving to the Full\u201d is more or less effective than treatment-as-usual. Because of the optimized active control condition, it is plausible that both treatments will result in a significant reduction in symptomology and related measures. In this scenario, the moderation and mediation analyses will still be of value in exploring if certain subgroups of patients benefit from one of the interventions in particular and if the processes through which the treatments achieved their effects differ. This sort of knowledge can contribute to a more personalized treatment approach by taking into account moderating factors and an increased effectiveness of interventions by refining and intensifying those components that focus directly on the mediators . AnotherThe study also has some limitations. First, the online nature of the screening procedure and intervention might limit the study sample. It excludes people that are not proficient with computers. These people might share certain characteristics , which reduces the representativeness of the sample. Second, the study might encounter difficulties in recruiting enough participants. Recruiting older adults for study participation is challenging . FurtherTo conclude, the proposed study will evaluate an ACT online self-help program combined with face-to-face contacts with the mental health counselor at the general practice for older adults with anxiety symptoms. Since subclinical anxiety is highly prevalent in this age group, (cost-)effective, low-threshold interventions are needed. The proposed study complies with this request. When \u201cLiving to the Full\u201d proves to be effective for this patient group, implementation of the intervention in general practices in the Netherlands will follow.Patient recruitment started November 2017. Recruitment is expected to be completed December 2018.Additional file 1:Spirit Checklist. (DOC 121 kb)Additional file 2:Spirit Figure. (PDF 118 kb)"}
+{"text": "Nanogold is widely used in many areas of physics and chemistry due to its environment-sensitive plasmon resonance absorption. The immobilization of gold nanoparticles in highly porous silica aerogel offers an attractive alternative to liquid gold solutions as they show a mechanically stable structure, are permeable to gases, and can even be used at elevated temperatures. We have found that the commercially available citrate-stabilized 10 nm gold nanoparticles may suffer from aggregation prior to or under the base-catalyzed gelation process of tetramethoxy silane. In the wet gels, Au particles increased in size, changed shape, and demonstrated the loss of plasmon resonance absorption, due to the formation of larger aggregates. We have studied a range of water-miscible organic solvents, stabilizing agents, and the gelation conditions to minimize changes from occurring in the aerogel setting and the supercritical drying process. It has been found that atmospheric carbon dioxide has a significant effect on aggregation, and it cannot be entirely excluded under normal synthetic conditions. Methanol resulted in an increase in the particle size only, while dimethyl sulfoxide, dimethylformamide, and urea changed the shape of nanoparticles to rod-like shapes, and diols led to an increase in both size and shape. However, using the polymeric stabilizer poly(vinyl pyrrolidone) efficiently prevented the aggregation of the particles, even in the presence of high concentrations of carbon dioxide, and allowed the production of nanoAu containing silica aerogels in a single step, without the modification of technology. When dispersed down to the few nanometer sizes, gold is not a \u201cnoble\u201d metal anymore. Nanogold particles (AuNPs) were extensively studied in the last two decades in several fields of science including physics, chemistry, biology, and medicine, leading to a sort of science \u201cgold rush\u201d . Due to 4 under a reductive environment that is provided by reducing agents like citric acid or NaBH4 [Gold nanoparticles are most commonly synthesized from tetrachloroauric acid HAuClor NaBH4 ,10,11,12or NaBH4 . The preor NaBH4 ,15.AuNPs of less than 5 nm in size are chemically reactive  and act Gold NPs are very important in chemical syntheses . HoweverAerogels are very low-density solids which keep the original structural characteristics of the wet gels that they are prepared from. They can be made of virtually any material that can be gelled ,31,32. TThe immobilization of gold NPs in silica aerogel offers the advantage of ease of handling and removal, as well as protection from aggregation by the steric hindrance of particle movements. Such aerogels can be used in several fields, including plasmonic sensing and nonlinear optical experiments, optical fiber detection, and catalytic reactions ,37.In this study, our aim was to synthesize nanogold-containing silica aerogel nanocomposites for gas sensing and catalytic applications. A solution of 10 nm gold nanoparticles stabilized with citrate ions, as well as 2 nm AuNPs stabilized with poly (PVA) was used as nanogold sources, respectively. Here, we report the difficulties that arose from the aggregation of the nanoparticles, the effects of solvents and environmental gases, and the use of stabilizing agents in order to prepare the required AuNP-silica aerogel composites.Silica aerogels were synthesized by the base-catalyzed sol-gel process, in which tetramethyl orthosilicate (TMOS) was hydrolyzed and condensed in a methanol-water mixture. In general, it is possible to mix particles or macromolecules in such a reaction mixture in order to create composites or hybrid aerogel materials, respectively ,39. HoweThe color change was due to the increased particle size of the AuNPs, as the plasmon resonance frequency is strongly correlated with the size and the shape of the nanogold particles . In an a2, and CO2) for their aggregation-inducing power. Carefully vacuumed and argon-filled Schlenk-type glasswares were used in the studies, in which diethanolamine was used as the organic solvent. Since DEA can reversibly dissolve a significant amount of carbon dioxide, we heated it up to 100 \u00b0C and purged it with argon to remove all of the adsorbed gases before the experiments. After filling the vessels with the Au solutions, their gas phase was purged with the selected gas, and the vessels were then sealed hermetically. We have tested three atmospheric gases  resulted in the highest degree of aggregation.Three kinds of behavior were observed. In methanol a a signiWe have tested different stabilizing agents to prevent the aggregation of the AuNPs in the aerogel reaction mixture. Poly (PVA), poly(ethylene glycol) (PEG), and poly(vinyl pyrrolidone) (PVP) were tested as polymeric agents. In addition, mercaptopropionic acid (MPA) and 3-mercaptopropyl trimethoxysilane (MPTMOS) were also tested due to the high affinity of gold NPs to sulfur donor atoms. PEG was sorted out in the process due to its low solubility in the TMOS hydrolysis mixture. For the stability tests, i-propanol, and EG was used as an aggregation-inducing solvent against which the stabilizing agents had to prove their power. MPTMOS was used in low concentration to create core-shell type particles. However, when mixed in the nanogold solution, it formed a precipitate in the solution. MPA was unable to prevent aggregation. PVA and PVP were both selected and tested in the real TMOS hydrolysis mixtures. Although they showed comparable activity, the dissolution of PVA in water took much longer, and it occasionally formed a precipitate in the reaction mixture with hydrolizing TMOS. As the stabilizing agent of choice, PVP proved to be the most advantageous among the tested ones, as it dissolved very rapidly, was compatible with the components of the reaction mixture, and prevented the aggregation of AuNPs until gelation occurred.3-water-methanol mixtures, it was possible to reproducibly synthesize multi-centimeter sized monolithic silica aerogel-gold nanocomposites without any aggregation of the nanogold guest particles.It has been found that the aggregation-free and reproducible synthesis of plasmonic silica aerogels containing small gold nanoparticles is a hard-to-control process. The system may behave differently, depending on the type of reaction vessel, the nature of the solvents used in the process, as well as the composition of the environmental gases. Atmospheric carbon dioxide has a strong negative impact, and solvents like n-propanol may initialize aggregation, even without carbon dioxide. After testing several stabilizing agents, poly(vinly pirrolidone) proved to be the most advantageous one. By using PVP in the TMOS-NHThe following chemicals and reagents were purchased and used without further purification. 10 nm Nanogold solution stabilized with citrate , 3-mercaptopropyl trimethoxysilane (MPTMOS) , Diethanolamine , ammonia solution of 25% , tetramethoxysilane (TMOS) , methanol , methanol , acetone , 2-propanol , 1-propanol , propylene glycol , ethylene glycol , ethanol , and poly(vinylpyrrolidone) .Photometry studies were performed with a Metertech SP-8001 spectrophotometer  in the 400\u20131100 nm range, using disposable PMMA cuvettes. Data were registered and analyzed by the Metertech UV-Mate software.2 drying of alcogels to aerogels were performed in a custom\u2013made high pressure reactor, according to the process described in the literature [Alcogels were prepared in plastic molds, were aged, and solvent was exchanged in aluminum drying frames. Extractions and supercritical COterature .2O, and 1.80 mL of aqueous ammonia solution (conc.: 25%). Solution A was magnetically stirred and solution B was mixed in rapidly. After 1 min of intensive stirring, the reaction mixture was poured into a 28 mm diameter cylindrical PVC plastic mold, which was lined with a thin poly(tetrafluoro ethylene) (PTFE) layer inside. The mold was covered with a double layer of parafilm to prevent evaporation. After one day of aging, the alcogel was transferred into a pitted aluminum frame and was soaked in methanol, methanol-acetone mixture, acetone, and copious amounts of freshly distilled dry acetone for 3 days to remove the water residues from the gel. After that, the wet gels were transferred into the high pressure tank reactor and were dried under supercritical conditions.Aerogel SH1 was synthesized by the following method. First, two solutions (A and B) were prepared before the reaction. Solution A was made of 16.0 mL of MeOH and 4.00 mL of TMOS. Solution B contained 16.0 mL of MeOH, 3.60 mL of HAerogel SH24 was synthesized according to the following procedure: 3.00 mL of 10 nm gold solution and 2.40 mL of aqueous PVP solution (conc.: 10%) were combined, and then 15.0 mL of MeOH and 3.00 mL of TMOS were added. A 1:10 volume ratio dilution from a 25% ammonia solution was prepared, and 2.70 mL of that was added to the reaction mixture. The mixture was poured in a plastic mold, and then the process described for SH1 was followed. Aerogel SH25 was prepared by the method described for SH24 on a 50% larger scale. Aerogel SH26 was prepared by the method described for SH25, except that 2 nm PVA-stabilized nanogold solution was used. Photographs of the as-prepared aerogels SH1, and SH24-SH26 are shown in Water-miscible solvents that are compatible with the general sol-gel procedure were tested to compare their effect on nanogold particles\u2019 aggregation. Two series of experiments were performed\u2014one in open cuvettes, and the other in hermetically sealed sample vessels. Spectral changes were monitored and recorded in the first three hours, and then daily for five days. Each solution was a 1:1 volumetric dilution of the commercially available nanogold solution, with the solvent of choice."}
+{"text": "Communications Biology 10.1038/s42003-020-0977-2, published online 21 May 2020.Correction to: In the original published version of the article, an error was introduced into Fig. 1a during the typesetting process. The error has been corrected in the PDF and HTML versions of the paper."}
+{"text": "In addition to metal chelation, bifunctional chelators have also been found to impact imaging outcomes due to their differences in stability, charge, hydrophilicity, etc. In the present work, a comparative pharmacokinetic evaluation and imaging characteristics were performed between 68Ga-labelled somatostatin analogues (TATE) using NOTA  and DOTA  as bifunctional chelating agents (BFCAs).68Ga-NOTA-TATE and 68Ga-DOTA-TATE were obtained with high radiochemical purity. 68Ga-NOTA-TATE demonstrated higher in vitro stability (\u2265 99%) than 68Ga-DOTA-TATE (\u2265 95%) after 3\u2009h of incubation. The water solubilities  and plasma protein binding rates (12.12% vs. 30.6%) were lower for 68Ga-NOTA-TATE than for 68Ga-DOTA-TATE. Differential pharmacokinetics and comparable tumour affinities (within 1\u2009h) were observed in AR42J tumour-bearing mice. Healthy volunteer imaging studies showed comparable distribution patterns of these two imaging agents. However, the maximum standardized uptake values (SUVmax) of the two tracers varied in each organ. The two PET agents demonstrated almost identical SUVmax values in the kidneys. 68Ga-NOTA-TATE did have a lower SUVmax in most other organs compared with 68Ga-DOTA-TATE, including the liver (4.2 vs. 10.1), potentially due to the lower protein binding rate.Both 68Ga-NOTA-TATE and 68Ga-DOTA-TATE demonstrated comparable tumour uptake in an AR42J mouse model. An initial clinical study revealed that 68Ga-NOTA-TATE may have reduced background uptake in the major organs such as the liver. Although the subject numbers were limited, further investigation of 68Ga-NOTA-TATE is warranted for detecting SSTR2-positive neuroendocrine tumours. Throl resin was mixed with 4 equivalents of each Fmoc protected amino acid and 3.9 equivalents of HATU (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolopyridinium 3-oxid hexafluorophosphate) as the coupling reagent in DMF. After incubation for 4\u2009h, the resin was washed with DMF 3 times and added to a mixture of 96:2:2 DMF/piperidine/DBU (v/v/v) for Fmoc deprotection. After 5\u2009min of incubation, the solvent was removed, and the coupling step was repeated sequentially according to the desired peptide sequence. The peptide was cleaved from the resin with a CH305, was 68Ga solution (10\u201315\u2009mCi/mL) and sodium acetate buffer (pH = 5.5) were prepared. The effects of the corresponding conjugate concentration, 68Ga activity, pH, temperature and reaction time on the radiochemical yield and purity of 68Ga-labelled NOTA-TATE and DOTA-TATE were investigated by independent reaction conditions. For comparing the distribution and imaging experiments of 68Ga-NOTA-TATE and 68Ga-DOTA-TATE, both were obtained under the following conditions: The amount of NOTA-TATE or DOTA-TATE was fixed at 20\u2009\u03bcg, and the activity of 68GaCl3 varied (370\u2009MBq-555\u2009MBq in 1\u2009mL of 0.05\u2009M HCl). The pH of respective mixtures was adjusted by adding sodium acetate buffer. The pH of the mixture was adjusted to 3.5\u20134, and 68Ga-NOTA-TATE was obtained by incubating at 90\u2009\u00b0C for 10\u2009min. 68Ga-DOTA-TATE was obtained by adjusting the pH of the mixture to 4\u20134.5 and incubating at 90\u2009\u00b0C for 10\u2009min. The radiolabelled conjugates were purified using preconditioned C-18 reversed-phase Sep-Pak cartridges.In this experiment, NOTA-TATE and DOTA-TATE solutions (20\u2009\u03bcg/10\u2009\u03bcL), 68Ga from the 68Ga complexes. The elution was monitored by detecting the radioactivity signal using an NaI (Tl) detector and the UV signal at 254\u2009nm. The flow rate was maintained at 1\u2009mL/min for elution.Characterization of the radiolabelled complexes was carried out using reversed-phase HPLC. Water (A) and acetonitrile (B) each mixed with 0.1% trifluoroacetic acid was used as the mobile phase with gradient elution  to separate the free 68Ga-labelled NOTA-TATE and DOTA-TATE were carried out by following a generalized procedure summarized below. One millilitre of fresh anticoagulated (heparinized) human plasma was prepared. Freshly labelled compound  was added to three centrifuge tubes, each containing 0.1\u2009mL of human plasma. After incubation at 37\u2009\u00b0C for 2\u2009h, 1\u2009mL of ice-cold methanol was added to each tube. The mixtures were separated by centrifugation  to collect the supernatant, which was repeated three times. A gamma counter was then used to measure the radioactivity of the supernatant (A) and the precipitate (B) in counts per minute (CPM); the plasma-protein binding rate was calculated as PPB = B/(A + B)100%, and the average of three tubes was recorded.In vitro serum binding studies for both To evaluate the in vitro stability, the radiolabelled preparation was incubated with human blood serum in a water bath at a constant temperature of 37\u2009\u00b0C. Aliquots (100\u2009\u03bcL) were taken from the reaction mixture at 15\u2009min, 30\u2009min, 1\u2009h, 2\u2009h, 3\u2009h and 4\u2009h, followed by precipitation of serum proteins using acetonitrile. The solution mixture was centrifuged, and the supernatant was analysed by HPLC using the protocol mentioned above. In addition, we added the labelled formulations to physiological saline and incubated them at 37\u2009\u00b0C. All the labelled compounds were tested by HPLC for radiochemical purity at each of the abovementioned time points.68Ga-labelled complexes was determined in the octanol water system by following the procedure mentioned below. In brief, 490\u2009\u03bcL of ultrapure water was added to each of three centrifuge tubes containing 0.5\u2009mL of saturated n-octanol and then combined with 10\u2009\u03bcL (1.85\u2009MBq/0.05\u2009mCi) of freshly prepared radiolabelled complexes by ultrasonication (3\u2009min). The centrifuge tube was allowed to stand for approximately 1\u2009min for the liquid to become stratified. The upper and lower liquids were defined as groups A  and B , accounting for the organic and aqueous phases, respectively. Then, 0.1\u2009mL was retrieved from each of the 6 sections. The radioactivity (CPM) of both phases was determined using a gamma counter, and the lipid-water partition coefficient was calculated as follows: logP = log (A-background)/(B-background). The average of three tubes was recorded as the logP.The lipid-water partition (logP) of 3.Tumour cells were subcutaneously injected into the left forelimb of each nude mouse. Biodistribution and imaging studies were conducted when the tumour volume reached at least 1.0 \u00d7 1.0 \u00d7 1.0\u2009cm68Ga-NOTA-TATE in AR42J tumour-bearing mice  was studied. Each mouse was injected with 0.1\u2009mL of 68Ga-NOTA-TATE  in the tail vein. At 15\u2009min, 30\u2009min, 1\u2009h and 2\u2009h after injection, groups of four tumour-bearing mice  were sacrificed by cervical dislocation to determine the percent injected dose per gramme (%ID/g) in various samples of mouse tissues  with a gamma counter. For comparison, biodistribution studies of 68Ga-DOTA-TATE were performed the same way.The biodistribution of 68Ga-NOTA-TATE or 68Ga-DOTA-TATE at a dose of 2.96\u2009MBq (0.08\u2009mCi). Images were acquired and reconstructed using micro-PET/CT at 30\u2009min and 2\u2009h after intravenous injection.Six  mice with AR42J tumour xenografts (15\u201320\u2009g) were used in the imaging study. All tumour-bearing mice were anaesthetized by intranasal inhalation of isoflurane. Each mouse was immediately injected with 0.1\u2009mL of 68Ga- NOTA-TATE low-dose group (100\u2009\u03bcCi) and a high-dose group (1\u2009mCi). Each group contained 10 mice, half female and half male. The dose of the appropriate compound was administered through the tail vein at an administration volume of 0.2\u2009mL. The animal blood routine, body weight and animal feed consumption were performed before administration and on days 4, 7, 14, 21 and 28 after administration. At the end of the observation period, the surviving mice were dissected, and histopathological examination was performed on the myocardium, liver, spleen, lung, kidney, stomach, intestine, gonad, bone, muscle and brain of each mouse.Thirty KM mice were randomly divided into a control group, a 68Ga-NOTA-TATE PET/CT scans, and ten volunteers  underwent 68Ga-DOTA-TATE PET/CT scans. All volunteers did not have a history of treatment with cold octreotide and did not fast before imaging. Each patient was injected with an average activity of 1.9\u2009MBq/kg. The timing of image acquisition ranged from 30 to 45\u2009min after injection (3\u2009min/bed position). The PET image was attenuated and then reconstructed using the iterative reconstruction algorithm implemented.Twenty-two healthy volunteers gave informed consent and agreed to participate in the study. Twelve volunteers  underwent 68GaCl3 and the amount of NOTA-TATE and DOTA-TATE on the labelling rate are shown in Fig. 68Ga-labelled NOTA-TATE  and DOTA-TATE . Under the optimal pH conditions, 68Ga-NOTA-TATE can be prepared at a lower temperature and in a shorter time. The molar activities of 68Ga-NOTA-TATE and 68Ga-DOTA-TATE obtained by labelling were 8.8\u2009GBq/mg and 7.4\u2009GBq/mg, respectively. HPLC analysis of the two complexes showed \u2265 99.0% radiochemical purity. The retention time of 68Ga-NOTA-TATE in the optimized HPLC system was 12.1 \u00b1 0.05\u2009min (n = 12), and the retention time of 68Ga-DOTA-TATE in the same gradient solvent system was 11.8 \u00b1 0.08\u2009min (n = 10) .The log P68Ga-NOTA-TATE and 68Ga-DOTA-TATE in nude mice bearing AR42J tumours is shown in Table 68Ga-NOTA-TATE in the gallbladder was significantly higher than that of 68Ga-DOTA-TATE, suggesting that 68Ga-NOTA-TATE can be excreted through the biliary system in mice. The radioactivity of 68Ga-NOTA-TATE in the spleen was significantly lower than that of 68Ga-DOTA-TATE. In animal experiments, the liver showed no significant difference in radioactivity uptake. Both complexes had high kidney uptake and low uptake in the bones and muscles. In the lungs, stomach and intestines, the activity of 68Ga-NOTA-TATE was slightly lower than that of 68Ga-DOTA-TATE. The radioactivity in the blood cleared quickly with time. After 1\u2009h of intravenous injection, the blood activity of 68Ga-NOTA-TATE and 68Ga-DOTA-TATE was only 0.58 \u00b1 0.24% ID/g and 0.35 \u00b1 0.20% ID/g, respectively. The tumour tissue had prominent uptake of both octreotide complexes. After intravenous injection of 68Ga-NOTA-TATE and 68Ga-DOTA-TATE, there was no significant difference in the radioactivity counts of the two compounds in tumour tissues: 15\u2009min (3.24 \u00b1 1.03% ID/g vs. 3.13 \u00b1 1.75% ID/g), 30\u2009min (3.97 \u00b1 2.24% ID/g vs. 3.62 \u00b1 1.94% ID/g) and 1\u2009h (3.16 \u00b1 1.92% ID/g vs. 3.14 \u00b1 2.07% ID/g), respectively. After 2\u2009h, the radioactivity count of 68Ga-NOTA-TATE (1.33 \u00b1 0.96% ID/g) in tumour tissue was lower than that of 68Ga-DOTA-TATE (2.3 \u00b1 1.34% ID/g).The biodistribution of 68Ga-NOTA-TATE imaging showed that the gallbladder had intense radioactivity, while 68Ga-DOTA-TATE imaging showed minimal gallbladder activity.Micro-PET/CT imaging of the AR42J tumour-bearing mice is shown in Fig. 68Ga-NOTA-TATE was injected into the tail vein of mice, the body weights of the animals in each administration group were not significantly different from that of the control group (P > 0.05), and the trend of feed consumption was comparable. The results of histopathological examination showed that the agent had no toxic pathological changes associated with the test substance. The WBC, PLT and RBC counts were all within the normal range.After 68Ga-NOTA-TATE (n = 12) and 68Ga-DOTA-TATE (n = 10) in the major organs of healthy volunteers are shown in Table 68Ga-DOTA-TATE activity in vivo. In the head and neck region, we observed that 68Ga-DOTA-TATE was not taken up in the brain. It should be noted that 68Ga-DOTA-TATE can be taken up by the pituitary gland (SUVmax = 7.9). Generally, 68Ga-DOTA-TATE can be diffusely taken up by the thyroid gland over a wide range (SUVmax = 1.5\u20135.9). In the chest region, we found that the level of 68Ga-DOTA-TATE uptake in lung tissue was low (SUVmax = 1.3), suggesting that the expression of SSTR2 in lung tissue was low. In the breast, 68Ga-DOTA-TATE mainly showed diffuse low uptake in glandular tissues (SUVmax = 0.4\u20130.9). In the abdominal region, we observed strong uptake of 68Ga-DOTA-TATE in the spleen (SUVmax = 33.5), which is related to the high expression of SSTR2 in splenic tissue. Diffuse high uptake of 68Ga-DOTA-TATE (SUVmax = 7.8\u201312.0) was observed in the liver. No radioactivity was seen in the gallbladder, indicating that 68Ga-DOTA-TATE is not excreted through the biliary system. 68Ga-DOTA-TATE was taken up in the pancreatic tissue at a moderate level, with a large range (SUVmax = 3.1\u20138.3), and the distribution was not uniform. 68Ga-DOTA-TATE was taken up at a moderate to high level in the gastrointestinal tract, and 68Ga-DOTA-TATE was relatively higher taken up by the stomach wall (SUVmax = 8.3). The adrenal glands could significantly take up 68Ga-DOTA-TATE (SUVmax = 17.7). 68Ga-DOTA-TATE is a hydrophilic compound that is mainly excreted by the urinary system. The distal renal tubules and collecting ducts of normal kidneys highly express somatostatin receptors, so both kidneys showed high radioactive activity (SUVmax = 17.8). Lymph nodes can take up 68Ga-DOTA-TATE (SUVmax = 0.5\u20132.0). The physiological distribution of 68Ga-NOTA-TATE in healthy volunteers is similar to that of 68Ga-DOTA-TATE. However, 68Ga-NOTA-TATE and 68Ga-DOTA-TATE have significant differences in the degree of uptake in some tissues and organs. 68Ga-NOTA-TATE demonstrated a lower average SUVmax in the pituitary gland (1.5 vs. 4.9), salivary glands (parotid gland (0.9 vs. 2.7), submandibular (1.2 vs. 3.4) and palatine tonsil (1.3 vs. 2.6)), thyroid (1.7 vs. 3.8), liver (4.2 vs. 10.1), spleen (14.6 vs. 26.9), pancreas (3.7 vs. 6.2), stomach (2.4 vs. 6.1), small bowel (1.8 vs. 3.4), large bowel (1.5 vs. 2.2) and adrenal glands (10.7 vs. 15.2). The average value of SUVmax in the blood pool was slightly higher for 68Ga-NOTA-TATE (1.4 vs. 0.9). The SUVmax of the kidneys (14.1 vs. 14.5) and other organs was comparable with that of 68Ga-DOTA-TATE from PET/CT imaging.Representative maximum-intensity projection images of the two radioactive compounds are shown in Fig. ATE n = 1 and 68Ga68Ga-DOTA-TATE plays an important role in NET staging, which often overexpresses SST2 [68Ga-based PET agents [68Ga complex adopts a pseudo-octahedral geometry with a log stability constant value of 21.33, as reported in the literature [68Ga, having a log stability constant value of 30.98 [68Ga complex not only with high thermodynamic stability but also with adequate kinetic inertness, especially in the pH range of 2\u20138 and in the presence of cations such as Ca2+, Mg2+ and Zn2+ found in blood serum [68Ga-NOTA conjugated somatostatin analogues will exhibit pharmacokinetic advantages over 68Ga-DOTA derivatives, and no direct comparison study of this rigour with clinical outputs has been reported. Therefore, in this report, we investigated the effect of BFCAs on the pharmacokinetics, tumour specificity and distribution of 68Ga-labelled somatostatin analogues in human subjects.With high affinity towards somatostatin receptor subtype 2 (SST2), ses SST2 . CompareT agents , 12. Theterature . In contof 30.98 . NOTA ca68Ga-DOTA-TATE, 68Ga-NOTA-TATE can be prepared (pH = 3.5) at a lower temperature (80\u2009\u00b0C) and with a shorter incubation time (5\u2009min). Both radiolabelled products have high radiochemical purity and good in vitro stability. The molar activity of 68Ga-NOTA-TATE (8.8\u2009GBq/mg) is higher than that of 68Ga-DOTA-TATE (7.4\u2009GBq/mg), which is one of the main advantages of NOTA tracers. It may also be a potential source of the differences observed in the PK values of the two tracers. NODAGA  is also a good dual-functional chelating agent for 68Ga. 68Ga-OPS202 (an SSTR antagonist), also called 68Ga-NODAGA-JR11 (JR11 = Cpa-c(DCys-Aph(Hor)-DAph(Cbm)-Lys-Thr-Cys)-DTyr-NH2), is a radiolabelled antagonist that may recognize more SST receptor binding sites [50) of 68Ga-OPS202 (antagonist) on SSTR2 is 1.2 \u00b1 0.2, and the half inhibitory concentration (IC50) of 68Ga-DOTA-TATE (agonist) on SSTR2 is 0.2 \u00b1 0.04, indicating that both have a high affinity for SSTR2 [68Ga-NOTA-TATE and 68Ga-DOTA-TATE, which may have a significant impact on the overall properties of each tracer. The 68Ga-NOTA complex is neutral, while the 68Ga-DOTA complex is negatively charged, which may facilitate faster clearance of the radiotracer through the urinary pathway from the physiological system. This may be the reason why 68Ga-labelled NOTA-TATE is less hydrophilic than 68Ga-DOTA-TATE and the clearance rate in the blood of tumour-bearing nude mice is also slightly lower. However, studies have shown that 68Ga-p-NH2-benzyl-NOTA has faster blood clearance and better pharmacokinetic properties than 68Ga-p-NH2-benzyl-DOTA [68Ga-labelled TATE prepared using either of these two BFCAs have comparable tumour affinities in nude mice bearing AR42J tumours. In micro-PET/CT imaging studies, the tumour could be clearly visualized, which correlates well with previous reports. Although the retention time of 68Ga-NOTA-TATE in tumour tissues seems to be slightly shorter than that of 68Ga-DOTA-TATE, the tumour uptake of the two is similar within 1\u2009h, and the tumour can still be clearly visualized after 2\u2009h. The animal experiments indicated that both DOTA and NOTA can be used as suitable BFCAs for preparing 68Ga-labelled TATE for PET imaging of neuroendocrine tumour lesions. The acute animal toxicity test of 68Ga-NOTA-TATE confirmed that it had no apparent toxicity.Compared to ng sites , 15 and or SSTR2 . The reazyl-DOTA . It can 68Ga-NOTA-TATE. To our knowledge, the distribution pattern of 68Ga-DOTA-TATE and the SUVmax in various organs has been documented [68Ga-NOTA-TATE was reported as the range of normal SUVmax in various organs, which was then compared with the SUVmax of 68Ga-DOTA-TATE accordingly. No apparent 68Ga-NOTA-TATE uptake was noted in the brain, which is similar to that of 68Ga-DOTA-TATE. However, in vitro studies found that somatostatin receptor 2 is expressed in the cerebral cortex, limbic system and basal ganglia [68Ga-NOTA-TATE, with a relatively low SUVmax. The study found that the expression of somatostatin receptor 2 (SSTR-2) in normal thyroid tissue and thyroid tumour tissue is different [68Ga-NOTA-TATE in the thyroid may have potential significance for the diagnosis of thyroid tumours. There was low 68Ga-NOTA-TATE uptake in the lungs, which may be because lung tissue mainly expresses somatostatin receptor 4 (SSTR4). In the blood pool, the SUVmax of 68Ga-NOTA-TATE is slightly higher than that of 68Ga-DOTA-TATE, which may be related to slower plasma clearance. In the breast, the observed uptake of 68Ga-NOTA-TATE was mostly in the glandular tissue and was diffuse and of low grade. In the abdomen, organs with a baseline expression of SSTR2  showed high 68Ga-NOTA-TATE uptake in vivo. The uneven spleen imaging pattern may be related to the anatomical distribution of the red and white pulp in the spleen parenchyma [68Ga-NOTA-TATE cannot be misdiagnosed as a tumour. Hepatocytes and hepatic stellate cells are negative for all five somatostatin receptor subtypes [68Ga-NOTA-TATE had intense radioactivity in the gallbladder of mice, but this was not seen in normal volunteer imaging. It is speculated that this may be due to the difference in the method of excretion of 68Ga-NOTA derivatives by mice and humans. The islets can be found in clusters in a pancreatic region since the SST2-positive subtype is located in the islets. Therefore, uneven accumulation of two tracers in the pancreatic region is a normal variant. The SUVmax of 68Ga-NOTA-TATE is lower in most other organs, especially in the liver, which is not reflected in the distribution experiments in animals. It is speculated that the reason for the lower SUVmax of 68Ga-NOTA-TATE in certain organs may be due to the lower protein binding rate or increased stability. This reduced background may increase the sensitivity towards primary endocrine tumours  and metastases (such as liver metastases). Both tracers are hydrophilic and are excreted through the urinary system. Moreover, the distal tubules and collecting ducts of normal kidneys highly express somatostatin receptors, so the bilateral kidneys show high radioactivity. We would like to point out that our study only involved normal volunteers. The observed low background of 68Ga-NOTA-TATE in normal organs warrants future evaluation of this agent in NET patients to detect SSTR2-positive lesions.All healthy volunteers showed no apparent side effects after the administration of cumented . In our  ganglia , suggestifferent . Thereforenchyma . Therefosubtypes . The upt68Ga. Under milder conditions, NOTA-TATE allows for rapid quantitative radiolabelling with higher radiochemical purity and serum stability compared with DOTA-TATE. Biodistribution in the AR42J tumour-bearing nude mice and micro-PET/CT imaging showed that 68Ga-NOTA-TATE and 68Ga-DOTA-TATE have comparable tumour uptake within 1\u2009h after injection. Healthy volunteer imaging studies showed similar distribution patterns, but the SUVmax of the two tracers varied in each organ. Except for the kidneys, the SUVmax of 68Ga-NOTA-TATE was lower than that of 68Ga-DOTA-TATE in most organs. This suggests a potential advantage of 68Ga-NOTA-TATE in the detection of primary and metastatic foci of neuroendocrine tumours due to the distinctively lower background. This study demonstrates that NOTA can be used in the preparation of 68Ga-labelled TATE and has potential for the diagnostic evaluation of neuroendocrine tumour lesions.In this study, NOTA-TATE was synthesized and radiolabelled with Additional file 1: Figure S1. LC-MS spectra for precursor NOTA-TATE.Additional file 2. Representative HPLC profiles of 68Ga-DOTA-TATE (A) and 68Ga-NOTA-TATE (B) with retention times of 11.8\u00b10.08 min and 12.1\u00b10.05 min, respectively."}
+{"text": "We aimed to analyze the distribution pattern of 68Ga-DOTA-TATE in normal subjects.Somatostatin is an endocrine peptide hormone that regulates neurotransmission and cell proliferation by interacting with G protein-coupled somatostatin receptors (SSTRs). SSTRs are specific molecular targets of several radiotracers for neuroendocrine tumor (NET) imaging. Gallium-68  without any biochemical, clinical, or imaging findings suggestive of NET were included in this study.A total of 617 consecutive 68Ga-DOTA-TATE was noted in the spleen followed by the kidneys, adrenal glands, liver, stomach, small intestine, prostate gland, pancreas head, pancreas body, thyroid gland, and uterus, in descending order. Minimal to mild uptake was detected in the submandibular glands, parotid glands, thymus, muscles, bones, breast, lungs, and mediastinum.The highest uptake of 68Ga-DOTA-TATE in normal subjects and the ranges of the maximum standard uptake value (SUVmax) and SUVmean values of 68Ga-DOTA-TATE obtained in several tissues for reliably identifying malignancy in 68Ga-DOTA-TATE PET/CT studies.Our study shows the biodistribution pattern of  Somatostatin is a peptide hormone that controls neurotransmission, hormone secretion, and cell proliferation by binding to somatostatin receptors (SSTRs). SSTRs are G protein-coupled membrane receptors presented on the cell surface of neuroendocrine cells. Five such receptor subtypes have been defined in humans ,2. SSTRs68Ga)-DOTA-TOC (DOTA-Tyr3-octreotide), 68Ga-DOTA-NOC , and 68Ga-DOTA-TATE (DOTA-Tyr3-octreotate) bind with varying affinity to SSTRs, and 68Ga-DOTA-TATE has shown higher affinity for SSTR subtype 2 (SSTR2)  values in several organs and compares the results with previous reports. The main difference between this study and those previously reported is that our study population was proven to be clinically or pathologically disease-free before the examination and during follow-up.The objective of this study is to investigate the normal distribution pattern and physiological variants of 68Ga-DOTA-TATE PET/CT whole-body scans performed in our department from May 2015 through April 2020 on patients with known or suspected neuroendocrine malignancies, mostly to evaluate adrenal adenomas. One hundred eighteen subjects without a diagnosis of NET, with no tracer avid lesion of NET on 68Ga-DOTA-TATE PET/CT, and followed up for at least 6 months (average: 2-3 years) without any clinical, biochemical, or imaging evidence of NET were included in this study. Patients with a history or diagnosis of malignancy and younger than 18 years were excluded.We retrospectively analyzed 617 consecutive This study was performed with Marmara University Faculty of Medicine Research Ethics Committee review approval . All patients included in the study gave written informed consent before the examination.68Ga-DOTA-TATE was prepared on a fully automated system using a standardized labeling sequence. Briefly, a commercially available germanium-68 (68Ge)/68Ga generator  was eluted with 0.6\u00a0M hydrochloric acid. Effluent containing the 68Ga fraction was transmitted to the PS-H+ cartridge to concentrate and purify 68Ga from residual 68Ge. The\u00a0purified 68Ga was then eluted with 1.7 mL 5 M sodium chloride into the reaction vial. Twenty-five micrograms DOTA-TATE  was dissolved using 3 mL of 1.5 M HEPES buffer solution in the reaction vial. The reaction was performed at 100 \u00b0C for 8 minutes. A C-18 light cartridge was used to purify the 68Ga-DOTA-TATE. The purified\u00a068Ga-DOTA-TATE was eluted with 1 mL ethanol and 1 mL water solutions and passed into a sterile vial. Radiochemical purity was over 95% in all cases, based on high-performance liquid chromatography.The 68Ga-DOTA-TATE PET/CT scans were conducted using a hybrid PET/CT scanner  in the Nuclear Medicine Department. Iohexol  was used as the oral contrast agent. 68Ga-DOTA-TATE (2 MBq/kg) was administered intravenously. Whole-body images from skull base to mid-thigh were acquired 60\u00b110 minutes after the injection. A low-dose 16-slice multidetector CT scan  was used to screen the body from mid-thigh to the base of the skull. A standard whole-body PET scan was conducted in 3D mode with an acquisition time of 3 min per bed position (six to eight bed positions) scanning the exact area with the CT scan. PET images were reconstructed with and without correction for attenuation using an iterative algorithm. Next, a workstation  was used for processing and interpretation.All 68Ga-DOTA-TATE PET/CT images were analyzed by two nuclear medicine physicians. Maximum SUV (SUVmax) and SUVmean values were calculated from a volume of interest (ROI) applied in the transaxial attenuation-corrected PET slice. ROIs obtained on CT images were applied to PET images. SUVmax was defined as the SUVmax in the ROI. SUVmean was taken as the average SUV concentration in ROI. SUVmax and SUVmean were evaluated on axial images in 29 normal anatomical structures for each patient using at least 2cm circular ROIs, avoiding inclusion of any activity from adjacent organs , frequency, and range] were calculated on Microsoft Excel for Mac version 16.37 (Microsoft Corporation).max values were categorized as high, moderate, mild, and minimal in accordance with the study of Moradi et al.  were men, and 73 patients (61.9%) were women. The average age of the patients was 51.83 years . The SUVmax >8.98 g/mL, which is the 50th percentile of hepatic uptake) was also noted in the kidneys, adrenal glands, and liver, in descending order. Moderate uptake  was observed in the stomach, small intestine, prostate gland, pancreas head and body, thyroid gland, and uterus. Mild uptake  was revealed in the submandibular and parotid glands. Minimal uptake (average SUVmax <2 g/mL) of tracer was observed in the thymus (n=12), gluteal and trapezius muscles, femora, iliac crest, breast tissue, lungs, and mediastinum. No specific uptake  was seen in the subcutaneous fat tissue and brain tissue. The average SUVmax (\u00b1 SD), average SUVmean (\u00b1 SD), and range of uptake on 68Ga-DOTA-TATE PET/CT for all the organs considered are summarized in max and SUVmean, respectively.Maximum physiological uptake was detected in the spleen. In addition to the spleen, high physiological uptake . Since islets may be present in clusters in any area of the pancreas, increased 68Ga-DOTA-TATE activity in such a region can be a normal variant for the pancreas.Variable uptake of s region . Howevers region  proved t68Ga-DOTA-TATE was also found in the adrenal glands. The reason for this relatively high uptake is the presence of the five subtypes of SSTRs, mostly SSTR2, in the adrenal gland, which has been shown by Epelbaum et al.  tract. Previous studies have identified the SSTRs in the lymphoid tissue associated with the gut, myenteric, and submucosal plexus  to infer the range of normal SUV values of physiological thymic 68Ga-DOTA-TATE in normal subjects. The ranges of the SUVmax and SUVmean values of 68Ga-DOTA-TATE obtained in the various organs is important for reliably identifying malignancy in 68Ga-DOTA-TATE PET/CT studies.This study shows the biodistribution pattern of"}
+{"text": "The four-parameter logistic (4PL) model has recently attracted much interest in educational testing and psychological measurement. This paper develops a new Gibbs-slice sampling algorithm for estimating the 4PL model parameters in a fully Bayesian framework. Here, the Gibbs algorithm is employed to improve the sampling efficiency by using the conjugate prior distributions in updating asymptote parameters. A slice sampling algorithm is used to update the 2PL model parameters, which overcomes the dependence of the Metropolis\u2013Hastings algorithm on the proposal distribution (tuning parameters). In fact, the Gibbs-slice sampling algorithm not only improves the accuracy of parameter estimation, but also enhances sampling efficiency. Simulation studies are conducted to show the good performance of the proposed Gibbs-slice sampling algorithm and to investigate the impact of different choices of prior distribution on the accuracy of parameter estimation. Based on Markov chain Monte Carlo samples from the posterior distributions, the deviance information criterion and the logarithm of the pseudomarginal likelihood are considered to assess the model fittings. Moreover, a detailed analysis of PISA data is carried out to illustrate the proposed methodology. Over the past four decades, item response theory (IRT) models have been extensively used in educational testing and psychological measurement  model and the Rasch model Rasch, , as wellmirt . In this case, using WinBUGS to infer the model parameters may lead to biased estimates when the sample size (the number of examinees) is small and the prior distributions then play an important role. To solve the above problems in using WinBUGS, Loken and Rulison  parameter, and dj is the item upper asymptote parameter. The 4PL model reduces to the other models as special cases: dj = 1 gives the 3PL model, cj = 0 gives the 2PL model, and aj = 1 gives the 1PL model. Following Culpepper .Fx the mean population level of ability to zero and the variance population level of ability to one  and the discrimination parameter a in scale.In the present study, the main aim is to evaluate the accuracy of parameter estimation obtained by the slice sampling algorithm for different types of prior distributions. Therefore, the first of the above methods is used to eliminate the trade-offs between ability \u03b8 and the difficulty parameter The motivation for the slice sampling algorithm  given by t(x) \u221d li(x) that cannot be sampled directly, where \u03d5(x) is a known density from which samples can be easily drawn and li(x) are non-negative invertible functions, which do not have to be density functions. We introduce the auxiliary variables represented by the vector 1, \u2026, \u03b4N are mutually independent. The inequalities \u03b4i < li(x) are established, and the joint density can be written asSuppose that the simulated values are generated from a target density function A) takes the value 1 if A is true and the value 0 if A is false. If the auxiliary variables are integrated out, the marginal distribution t(x) is obtained aswhere the indicator function I(li(x), we can then obtain the set \u039bi\u03b4 = {x\u2223\u03b4i < li(x)}. The simulated values are generated from the Gibbs sampler based on the auxiliary variables by repeatedly sampling from the full conditional distributions, proceeding as follows at iteration r:Using the invertibility of the function i = 1, \u2026, N.Sample Sample We thereby derive a horizontal \u201cslice\u201d under the density function. Thus, a Markov chain based on the new Gibbs sampler can be constructed by sampling points alternately from the uniform distribution under the density curve and only concerning the horizontal \u201cslice\u201d defined by the current sample points.In the Bayesian framework, we first consider the benefits of the slice sampling algorithm compared with the traditional Metropolis\u2013Hastings (MH) algorithm  given by t(x) \u221d \u03d5(x)l(x), where \u03d5(x) is selected as a special proposal distribution. Let x* be a candidate value from the proposal distribution \u03d5(x) and let xr)/l(xr)}, is determined by a random number u from Uniform. Essentially, if u < l(x*)/l(xr), then xr+1)( = xr) such that u < l(x*)/l(xr). Therefore, x* can be regarded as a sample from \u03d5(x) restricted to the set Let us use the MH algorithm to obtain samples from the posterior distribution In addition, with the MH algorithm, it is relatively difficult to sample parameters with monotonicity or truncated interval restrictions. Instead, it is possible to improve the accuracy of parameter estimation by employing strong informative prior distributions to avoid violating the restriction conditions  for k = 1, \u2026, p, which is a realization of t(X). This involves sampling from \u03d5(xk\u2223xk)(\u2212) restricted to the set l(\u2212) is invertible for all k given xk), the non-informative priors NI(a > 0), and the inappropriate priors Exp(1) and Gamma.It is well-known that the Gibbs algorithm can quickly and effectively draw samples from the posterior distribution owing to the fact that the full conditional posterior distribution is easy to sample using the conjugate prior distribution. However, the Gibbs algorithm is not valid for Bayesian non-conjugate models such as the 2PL model. By comparison, the slice sampling algorithm for estimating the 2PL model has the advantage of a flexible prior distribution being introduced to obtain samples from the full conditional posterior distributions rather than being restricted to using the conjugate distributions, which is required in Gibbs sampling and is limited to the use of the normal ogive framework , we can obtain the full conditional distribution of \u03b7ij based on Bayes' theorem:First, following B\u00e9guin and Glas , we intrwherecj ~ Beta, \u03b3j ~ Beta. However, the guessing and slipping parameters themselves satisfy the following truncated restrictions owing to model identification  can be written asThe full conditional posterior distributions of /(1 \u2212 \u03b3j \u2212 cj) is the correct response probability of the 2PL model, while (1 \u2212 \u03b3j \u2212 Pij)/(1 \u2212 \u03b3j \u2212 cj) is the corresponding incorrect response probability. Therefore, the joint likelihood of a, b, c, \u03b3, \u03b8 based on the auxiliary variables \u03bb and \u03c6 can be written asare introduced to facilitate sampling, where Equivalently,whereIntegrating out the two random variables \u03bb and \u03c6 in (10), the joint likelihood based on responses can be obtained:E\u03bb is an expectation operation for the random variable \u03bb. We know that \u03b7, \u03bb, and \u03c6 are independent of each other. Therefore, the joint posterior distribution based on the auxiliary variables can be written aswhere The specific form can be represented asThe detailed slice sampling algorithm is given below.ij and \u03c6ij when given \u03b8i, aj, bj, cj, \u03b3j, and yij. According to (13), the auxiliary variables \u03bbij and \u03c6ij have the following interval constraints:First, we update the auxiliary variables \u03bbij and \u03c6ij can be written asTherefore, the full conditional posterior distributions of \u03bbbj. The prior of the difficulty parameter is assumed to follow a normal distribution with mean \u03bcb and variance i, when yij = 1, we have Next, we update the difficulty parameter In fact, this inequality is obtained through the following calculation process:from whichTherefore, we haveFinally, we obtain the following inequality:i, when yij = 0, we have In the same way, \u2200bj:Using the above inequalities i \u2212 (1/1.7aj)log[(1 \u2212 \u03c6ij)/\u03c6ij] among all the examinees who correctly answer the jth item. Similarly, we can obtain a minimum of \u03b8i \u2212 (1/1.7aj)log[\u03bbij/(1 \u2212 \u03bbij)] among all the examinees who mistakenly answer the jth item. Finally, the full conditional posterior distribution of bj can be obtained as a truncated prior distribution, with the truncated interval between maximum and minimum. The specific mathematical expressions are as follows.If this truncated interval is narrow, the sampling efficiency is improved and the parameter can converge fast. Therefore, we need to limit the upper and lower bounds of the truncated interval. In fact, we can obtain a maximum of \u03b8aj, cj, \u03b3j, \u03b8, \u03bb, \u03c6, and y, the full conditional posterior distribution of bj isLet whereaj. To ensure that this parameter is greater than zero, we use a truncated normal distribution with mean \u03bca and variance yij = 1, \u2200i, \u03b8i \u2212 bj > 0, we have yij = 0, \u2200i, \u03b8i \u2212 bj < 0, we have aj can be established using a procedure similar to that used above to derive the truncated interval for the difficulty parameter bj:Subsequently, we update the discrimination parameter yij = 1, \u2200i, \u03b8i \u2212 bj < 0, we have yij = 0, \u2200i, \u03b8i \u2212 bj > 0, we have Similarly, when Letbj, cj, \u03b3j, \u03bb, \u03c6, \u03b8, and y, the full conditional posterior distribution of aj is given byGiven whereIn fact, the discrimination parameter is set to be greater than zero in the item response theory. Therefore, the prior distribution for the discrimination parameter is assumed to be a normal distribution truncated at 0. Based on the likelihood information, we can obtain the truncation interval of the discrimination parameter. However, the left endpoint of the truncation interval may be <0. In this case, we need to add 0 to the truncation interval to restrict the left endpoint in 17.i. The prior of \u03b8i is assumed to follow a normal distribution, i is sampled from the following normal distribution with truncated interval between Finally, we update the latent ability \u03b8where\u03a9 = , where \u03a9(1), \u2026, \u03a9R) and (16)\u2013(18), where i = 1, \u2026, N, j = 1, \u2026, J, and r = 1, \u2026, R. The joint likelihood function of the responses can be written asIn this paper, two Bayesian model assessment methods are considered to fit three different models , namely, the deviance information criterion  is the probability of response. The logarithm of the joint likelihood function in (19) evaluated at \u03a9r) = \u22122log L(Y \u2223 \u03a9), PD given in (21) are always non-negative. The model with a smaller DIC value fits the data better.In (21), Letting Note that the maximum value adjustment used in The model with a larger LPML has a better fit to the data.This simulation study is conducted to evaluate the recovery performance of the Gibbs-slice sampling algorithm based on different simulation conditions.J = 20 or 40 and (b) number of examinees N = 500, 1, 000, or 2, 000. Fully crossing different levels of these two factors yield six conditions (two test lengths \u00d7 three sample sizes). Next, the true values of the parameters are given. True values of the item discrimination parameters aj are generated from a uniform distribution, i.e., aj ~ U, j = 1, 2, \u2026, J. The item difficulty parameters bj are generated from a standardized normal distribution. The item guessing and slipping parameters  are generated from cj ~ U and \u03b3j ~ UI(\u03b3j < 1 \u2212 cj). The ability parameters of examinees \u03b8i are also generated from a standardized normal distribution. In addition, we adopt non-informative prior distributions for the item parameters, i.e., gj ~ Beta, and \u03b3j ~ Beta, j = 1, 2, \u2026, J. The prior for the ability parameters is assumed to follow a standardized normal distribution owing to the model identification restrictions. One hundred replications are considered for each simulation condition.The following manipulated conditions are considered: (a) test length To evaluate the convergence of parameter estimation, we only consider convergence in the case of minimum sample sizes owing to space limitations. That is, the test length is fixed at 20, and the number of examinees is 500. Two methods are used to check the convergence of our algorithm: the \u201ceyeball\u201d method to monitor convergence by visually inspecting the history plots of the generated sequences ((\u03b7) denote the posterior mean and the posterior standard deviation of \u03b7 obtained from the mth simulated data set for m = 1, \u2026, M. The Bias for the parameter \u03b7 is defined asThe accuracy of the parameter estimates is measured by four evaluation criteria, namely, the Bias, mean squared error (MSE), standard deviation (SD), and coverage probability (CP) of the 95% highest probability density interval (HPDI) statistics. Let \u03b7 be the parameter of interest. Assume that the MSE for \u03b7 is defined asand the average of the posterior standard deviation is defined asBias and MSE are important criteria used to evaluate the accuracy of parameter estimation in a simulation study. These criteria are used to investigate the relative distance between the parameter estimator and the true value. The greater the distance between the parameter estimator and the true value, the lower is the accuracy of parameter estimation and the poorer is the performance of the algorithm. However, for real data analysis, it is impossible to calculate Bias and MSE. The SD, on the other hand, can be calculated from the posterior samples of a Markov chain in simulation studies and real data analysis. In our simulation study, we calculate the average SD through repeated experiments to eliminate the error caused by randomness in a single simulation experiment.The coverage probability is defined asThe average Bias, MSE, SD, and CP for item parameters based on six different simulation conditions are shown in Given the total test length, when the number of individuals increases from 500 to 2,000, the average MSE and SD for discrimination, difficulty guessing, and slipping parameters show a decreasing trend. For example, let us consider a total test length of 20 items. When the number of examinees increases from 500 to 2,000, the average MSE and the average SD of all discrimination parameters decrease from 0.0625 to 0.0474 and from 0.1460 to 0.0759, respectively. The average MSE and the average SD of all difficulty parameters decrease from 0.0505 to 0.0263 and from 0.0559 to 0.0260, respectively. The average MSE and the average SD of all guessing parameters decrease from 0.0092 to 0.0023 and from 0.0247 to 0.0156, respectively. The average MSE and the average SD of all slipping parameters decrease from 0.0060 to 0.0025 and from 0.0260 to 0.0166, respectively.Under the six simulated conditions, the average CPs of the discrimination, difficulty guessing, and slipping parameters are about 0.9500.When the number of examinees is fixed at 500, 1,000, or 2,000, and the number of items is fixed at 40, the average MSE and SD show that the recovery results of the discrimination, difficulty, guessing, and slipping parameters are close to those in the case where the total test length is 20, which indicates that the Gibbs-slice sampling algorithm is stable and there is no reduction in accuracy owing to an increase in the number of items.In summary, the Gibbs-slice sampling algorithm provides accurate estimates of the item parameters in term of various numbers of examinees and items. Next, we will explain why the Bias criterion is useful, and why it seems irrelevant in the simulation study.If we want to determine whether our algorithm estimates the parameter accurately, we need more information to infer the parameter, which requires a large sample size. Here, Bias is an important criterion to evaluate the accuracy of parameter estimation. Let us give an example to illustrate the role of Bias. In Simulation Study 1, suppose that we investigate the accuracy of the algorithm in estimating a discrimination parameter. When the number of examinees increases from 500 to 2,000, the Bias of the discrimination parameter should show a decreasing trend. The result of Bias reduction further verifies that a greater number of samples are needed to improve the accuracy of parameter estimation.In Simulation Study 1, we cannot enumerate the Bias of each item parameter one by one because there are too many simulation conditions and we are subject to space limitations. Therefore, we choose to calculate the average Bias of the parameter of interest. Next, we take the discrimination parameters as an example to further explain why Bias seems irrelevant in Simulation Study 1. Suppose that we have obtained 40 Biases of discrimination parameters, that the Bias values of these 40 discrimination parameters are either positive or negative, and that the average Bias of all 40 items is close to 0. However, the near-zero value of the average Bias does not show whether the parameter estimation is accurate or the result is caused by the positive and negative superposition of the 40 Biases. In fact, we find that the Bias for each item discrimination parameter show a decreasing trend with increasing number of examinees. To sum up, we do not analyze the results of the average Bias in the simulation studies, but Bias is indeed an important criterion to evaluate the accuracy of each parameter estimation.Next, we evaluate the recovery of the latent ability using four accuracy evaluation criteria. The following conclusions can be obtained from Given a fixed number of examinees , when the number of items increases from 20 to 40, the average MSE and SD for the ability parameters also show a decreasing trend.Under the six simulated conditions, the average CP of the ability is also about 0.9500.Given a fixed number of examinees , when the number of items increases from 20 to 40, the correlation between the estimates and the true values tends to increase. For example, for 500 examinees, when the number of items increases from 20 to 40, the correlation between the estimates and the true values increases from 0.8631 to 0.9102.Given a fixed number of items (20 or 40), when the number of examinees increases from 500 to 2,000, the correlation between the estimates and the true values remains basically the same.In summary, it is shown again that the Gibbs-slice sampling algorithm is effective and that the estimated results are accurate under various simulation conditions.c ~ Beta and \u03b3 ~ Beta, and we focus on the influence of different prior distributions on the accuracy of parameter estimation in the process of implementing the slice sampling algorithm. Note that in this simulation study, we do not focus on the accuracy of the guessing and slipping parameters, since Culpepper  and person parameters, and to address the sensitivity of the algorithm with different priors. Three types of prior distributions are examined: informative priors, non-informative priors, and inappropriate priors.N = 1, 000, and the test length J = 20. The true values for the items and persons are the same as in Simulation Study 1. One hundred replications are considered for each simulation condition. The following three kinds of prior distributions are considered in implementing the slice sampling algorithm:The number of the examinees a ~ logN, b ~ N, and \u03b8 ~ N;informative prior: a ~ NI, b ~ Uniform, and \u03b8 ~ N;non-informative prior: a ~ Exp(1), b ~ t(1), and \u03b8 ~ t(1); (2) a ~ Gamma, b ~ Cauchy, and \u03b8 ~ Cauchy.inappropriate prior: (1) a and b based on the three kinds of prior distribution are shown in The Gibbs-slice sampling algorithm is iterated 20,000 times. The first 10,000 iterations are discarded as burn-in. The PSRF values of all parameters are <1.2. The Bias, MSE, and SD of a and b are almost the same under different prior distributions. This shows that accuracy of parameter estimation can be guaranteed by the slice sampling algorithm, no matter what prior distribution is chosen, as long as the values sampled from this distribution belong to a reasonable parameter support set. In addition, the Bias, MSE and SD of a and b fluctuate around 0, which shows that the slice sampling algorithm is accurate and effective in estimating the item parameters.From Next, we evaluate the recovery of the latent ability based on different prior distributions in In this simulation study, we use two Bayesian model assessment criteria to evaluate the model fittings. Two issues warrant further study. The first is whether the two criteria can accurately identify the true models under different design conditions. The second is that we study the phenomena of over-fitting and under-fitting between the true model and the fitting models.N = 1, 000 is considered and the test length is fixed at 40. Three item response models are considered: the 2PL, 3PL, and 4PL models. Thus, we evaluate model fitting in the following three cases:In this simulation, a number of individuals Case 1: 2PL model (true model) vs. 2PL model, 3PL model, or 4PL model (fitted model).Case 2: 3PL model (true model) vs. 2PL model, 3PL model, or 4PL model (fitted model).Case 3: 4PL model (true model) vs. 2PL model, 3PL model, or 4PL model (fitted model).The true values and prior distributions for the parameters are specified in the same way as in Simulation Study 1. To implement the MCMC sampling algorithm, chains of length 20,000 with an initial burn-in period of 10,000 are chosen. There are 100 replications for each simulation condition. The potential scale reduction factor  science data are used. Among the many countries that have participated in this computer-based assessment of sciences, we choose students from the USA as the object of analysis. The original sample size of students is 658, and 110 students with Not Reached  or Not Response  are removed, with Not Reached and Not Response (omitted) being treated as missing data. The final 548 students answer 16 items. All 16 items are scored using a dichotomous scale. The descriptive statistics for these PISA data are shown in We consider three models to fit the PISA data: the 2PL, 3PL, and 4PL models. In the estimation procedure, the same non-informative priors as in Simulation Study 1 are utilized for the unknown parameters. In all of the Bayesian computations, we use 20,000 MCMC samples after a burn-in of 10,000 iterations for each model to compute all posterior estimates. The convergence of the chains is checked by PSRF. The PSRF values of all item and ability parameters for each model are <1.2. On this basis, the results of Bayesian model assessment for the PISA data are shown in According to DIC and LPML in Next, we will use the 4PL model to analyze the PISA data in detail based on the results of the model assessment.The estimated results for the item parameters are shown in The histograms of the posterior estimates of the ability parameters are shown in In this paper, an efficient Gibbs-slice sampling algorithm in a fully Bayesian framework has been proposed to estimate the 4PL model. This algorithm, as its name suggests, can be conceived of as an extension of the Gibbs algorithm. The sampling process consists of two parts. One part is the Gibbs algorithm, which is used to update the guessing and slipping parameters when non-informative uniform priors are employed for cases that are prototypical of educational and psychopathology items. This part implements sampling by using a conjugate prior and greatly increases efficiency. The other part is the slice sampling algorithm, which samples the 2PL IRT model from the truncated full conditional posterior distribution by introducing auxiliary variables. The motivations for the slice sampling algorithm are manifold. First, this algorithm has the advantage of flexibility in the choice of prior distribution to obtain samples from the full conditional posterior distributions, rather than being restricted to using the conjugate distributions as in the Gibbs sampling process, which is also limited to the normal ogive framework. This allows the use of informative priors, non-informative priors, and inappropriate priors for the item parameters. Second, the Metropolis\u2013Hastings algorithm depends on the proposal distributions and variances (tuning parameters) and is sensitive to step size. If the step size is too small, the chain will take longer to traverse the target density. If the step size is too large, there will be inefficiencies due to a high rejection rate. However, the slice sampling algorithm can automatically tune the step size to match the local shape of the target density and draw samples with acceptance probability equal to one. Thus, it is easier and more efficient to implement.However, the computational burden of the Gibbs-slice sampling algorithm becomes intensive, especially when a large numbers of examinees or items are considered, or a large MCMC sample size is used. Therefore, it is desirable to develop a standalone R package associated with C++ or Fortran software for more a extensive large-scale assessment program. In fact, the new algorithm based on auxiliary variables can be extended to estimate some more complex item response and response time models, for example, the graded response model or the Weibull response time model. Only DIC and LPML have been considered in this study, but other Bayesian model selection criteria such as marginal likelihoods may also be potentially useful to compare different IRT models. These extensions are beyond the scope of this paper but are currently under further investigation.http://www.oecd.org/pisa/data/.Publicly available datasets were analyzed in this study. This data can be found here: JZ completed the writing of the article and provided article revisions. JL provided original thoughts. JZ, JL, HD, and ZZ provided key technical support. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "These additional properties have led to the hypothesis that carvedilol may improve cardiac contractility in the diabetic heart to a greater extent than metoprolol. The present study aimed to compare the efficacy of metoprolol and carvedilol on myocardial function in animal models and cardiac tissue from patients with type 2 diabetes and preserved ejection fraction.Increasing cohorts of patients present with diabetic cardiomyopathy, and with no targeted options, treatment often rely on generic pharmaceuticals such as \u03b2\u2010blockers. \u03b2\u2010blocker efficacy is heterogenous, with second generation \u03b2\u2010blocker metoprolol selectively inhibiting \u03b2Echocardiographic examination of cardiac function and assessment of myocardial function in isolated trabeculae was carried out in patients with and without diabetes undergoing coronary artery bypass grafting (CABG) who were prescribed metoprolol or carvedilol. Equivalent measures were undertaken in Zucker Diabetic Fatty (ZDF) rats following 4\u00a0weeks treatment with metoprolol or carvedilol.Patients receiving carvedilol compared to metoprolol had no difference in cardiac function, and no difference was apparent in myocardial function between \u03b2\u2010blockers. Both \u03b2\u2010blockers similarly improved myocardial function in diabetic ZDF rats treated for 4\u00a0weeks, without significantly affecting in vivo cardiac function.Metoprolol and carvedilol were found to have no effect on cardiac function in type 2 diabetes with preserved ejection fraction, and were similarly effective in preventing myocardial dysfunction in ZDF rats. In this manuscript, we present data comparing the effects of carvedilol and metoprolol on in vivo and in vitro cardiac function in both diabetic and non\u2010diabetic human patients, as well as in a rat model of type 2 diabetes (Zucker Diabetic Fatty rats). Our data demonstrate that both carvedilol and metoprolol are able to improve the contractility of cardiac muscle from diabetic humans and rats, as well as preserving cardiac function in both models. Importantly, we saw no extra improvement in individuals or animals that received carvedilol over those who received metoprolol, suggesting that any improvement in health outcomes provided by carvedilol (as seen in the COMET study) are not derived from differences in the contractile function of the cardiac muscle. Meanwhile, third generation \u03b2\u2010blockers, including carvedilol, nonselectively inhibit \u03b2\u2010ARs with additional inhibition of \u03b11\u2010ARs  inhibitors are commonly prescribed across a range of cardiac dysfunctions. All \u03b2\u2010blockers inhibit \u03b2\u2010AR activation, maintain calcium homeostasis in myocytes and prevent arrhythmia. However, there is a range of \u03b2\u2010blockers in use, and not all \u03b2\u2010blockers are equal  rats following \u03b2\u2010blocker treatment. This approach allowed us to determine whether metoprolol or carvedilol is a more effective \u03b2\u2010blocker to preserve cardiac function at the tissue and whole heart levels, as well as to test the hypothesis that carvedilol confers greater protection against mortality by improving contractility in the myocardium to a greater extent than metoprolol.22.1\u22121) or carvedilol (26.7\u00a0\u00b1\u00a03.0\u00a0mg\u00a0day\u22121) were grouped into those without (ND) and those with type 2 diabetes (DM), all of whom received clinical diagnosis at least 1\u00a0year prior to their surgery.The local Human Ethics Committee approved the study and all patients provided informed consent. Human epicardial right atrial appendages (RAA) were acquired from coronary artery disease patients with preserved ejection fraction who underwent on\u2010pump coronary artery bypass graft (CABG) surgery, prior to cardioplegia. Patients requiring emergency CABG, with ongoing myocardial ischemia prior to CABG or those with concomitant cardiac surgical procedures were excluded. All patients had a preoperative transthoracic echocardiogram and functional parameters were determined as previously described  with 0.5\u00a0mM Ca2+ and 6.25\u00a0mM 2,3\u2010butanedione monoxime (BDM) that had been well oxygenated with carbogen (95% O2:5% CO2). The RAA tissue was immediately returned to the laboratory, such that the dissection of tiny cardiac muscles (trabeculae) commenced 5\u201310\u00a0min after removal. Freshly dissected trabeculae were transferred to an experimental bath and attached between a force transducer and a micromanipulator. Basal and length dependency of force development was measured in the isolated trabeculae, as previously described . To impose similar stretch levels the muscles in both groups were stretched to the length (Lmax) at which isometric developed force (Fdev) was maximal. Basal Fdev was determined following a 1\u2010hr equilibration.Myocardial function was assessed in a subset of patients for whom cardiac tissue was available. RAAs were removed under normothermic conditions before cross clamping for cardiopulmonary bypass. Immediately after removal, all specimens were placed in a sealed vial containing a modified Krebs\u2013Henseleit buffer (KHB) . Zucker Diabetic Fatty (ZDF) rats have a homozygous missense mutation in the leptin receptor gene (fa/fa) leading to impaired satiety signaling and hyperphagia .At the completion of the 4\u2010week treatment, rats were anesthetized with isoflurane  for blood sampling and echocardiography. Blood was sampled from the tail vein for immediate determination of plasma glucose concentrations using a glucometer . The upper detection limit of 33.33\u00a0mmol\u00a0L2.4Anesthetized rats underwent a transthoracic echocardiogram, using a Vivid E9 ultrasound system . Standard two\u2010dimensional echocardiographic left ventricular parameters were obtained from the parasternal short axis, along with pulsed Doppler images of the mitral valve inflow to estimate diastolic function (E/A ratio). Animals were allowed to recover for 1\u20132\u00a0days after echocardiography, prior to terminal procedures.2.5\u22121), and placed in a modified KHB  with 0.5 CaCl2 and 18.5 KCl, and oxygenated with carbogen (95% O2:5% CO2). The hearts were retrograde perfused via the aorta while cardiac trabeculae were dissected from the right ventricle , mounted on a force transducer, and prepared at Lmax as described above . After an equilibration period of 20\u00a0min, force\u2013frequency relationships were obtained by measuring steady\u2010state twitch force conditions at stimulation frequencies of 2, 3, 4, 5, and 6\u00a0Hz.Hearts were rapidly extracted from rats anesthetized with pentobarbital . Force values were normalized to the cross\u2010sectional area of the trabeculae (width\u00a0\u00d7\u00a0thickness\u00a0\u00d7\u00a0\u03c0). Statistical analyses were conducted with GraphPad Prism (version 7). Differences amongst groups were compared via a two\u2010way ANOVA followed by a Holm\u2013Sidak post hoc test, except for \u03b2\u2010blockade intake which was assessed by 33.1Patients with diabetes undergoing CABG surgery had higher glucose and HbA1c levels compared to patients without diabetes, in line with their diagnosis (Table\u00a03.2p\u00a0=\u00a0.052), suggestive of impaired diastolic function although all values were within the normal clinical range.Presurgical echocardiographic assessment was clinically indicated for all patients. Analysis of these data indicated mild systolic dysfunction in most groups, with values for ejection fraction at or slightly below 50% Figure\u00a0. Most otOverall, patients prescribed carvedilol exhibited increased left ventricular internal diameter during both systole and diastole Figure\u00a0, indicatTaken together, these data suggest that cardiac function is compromised in all patients, unsurprising for a cohort of patients undergoing a CABG procedure, and that patients with diabetes exhibit increased diastolic dysfunction. Patients, both DM and ND, prescribed carvedilol exhibited similar contractile performance to those prescribed metoprolol, with a mild reduction in ejection fraction and fractional shortening in the ND group.3.3dev and maximal rate of contraction  compared to patients without diabetes. However, there was no significant difference in any parameter of myocardial function in trabeculae from patients with type 2 diabetes prescribed carvedilol compared to metoprolol. This observation was not consistent with our hypothesis that patients receiving carvedilol would have greater myocardial contractility than patients receiving metoprolol.Myocardial function was assessed in the trabeculae isolated from the right atrial appendage of patients undergoing coronary artery bypass graft surgery Figure\u00a0. Trabecun Figure\u00a0,c, and a3.4Our experiments in trabeculae from human patients indicated no differential effects of carvedilol and metoprolol on myocardial and whole heart function in type 2 diabetes. However, these data could not address the ability of the two \u03b2\u2010blockers to preserve cardiac function, as ethical patient care precludes including a group with no intervention. Moreover, all human tissue used in this study was donated by patients undergoing CABG surgery, precluding a healthy control for comparison. Thus, we repeated our experiments in a ZDF rat model of type 2 diabetes.Basal characteristics of ZDF rats were assessed in 20\u2010week old animals following 4\u2010week treatment with metoprolol, carvedilol, or control diet Table\u00a0, a good Food intake was monitored, and calculated per rat per day Table\u00a0. Rats wi3.5\u22121, p\u00a0<\u00a0.05 ND vs. DM). This contributed to an increased E/A ratio in DM, suggestive of diastolic impairment, although values remained in the normal range . Taken together, these data demonstrate that cardiac function in the 20\u2010week ZDF rats was mildly impaired compared to lean littermates, similar to our human cohort.The effects of diabetes and \u03b2\u2010blockade on in vivo cardiac function were assessed by echocardiography Figure\u00a0. DM ratse Figure\u00a0. Furtherp\u00a0<\u00a0.05 ND metoprolol vs. ND control). Thus, while diabetes led to mild systolic and diastolic impairments, there was no effect of either carvedilol or metoprolol on cardiac function at this early time point.Chronic \u03b2\u2010blockade had little impact on in vivo cardiac function, with only a further reduction in heart rate in DM animals treated with metoprolol Figure\u00a0, and inc3.6dev; Figure\u00a0max; Figure\u00a0min; Figure\u00a0To assess the myocardial effects of \u03b2\u2010blockade, trabeculae were isolated from the right ventricle of ZDF rat hearts following 4\u2010week treatment with metoprolol, carvedilol, or control diet Figure\u00a0. Type 2 max; Figure\u00a0While metoprolol and carvedilol improved myocardial function in diabetes, chronic \u03b2\u2010blockade in ND animals did not increase ex vivo contractility or relaxation Figure\u00a0. Indeed,4A lack of suitable, specific treatments hampers outcomes for patients with diabetic cardiomyopathy. It has been suggested that within \u03b2\u2010blocker treatments, carvedilol elicits better outcomes than metoprolol in heart failure, with additional benefits that may extend to type 2 diabetes Sica, . We hypoAlthough we did not identify differential activity of carvedilol and metoprolol, both \u03b2\u2010blockers improved myocardial function compared to untreated type 2 diabetic rats, without an effect on in vivo cardiac function. Our data do therefore suggest that a critical positive effect of \u03b2\u2010blockers in the diabetic heart is the preservation of contractile function, as demonstrated in the controlled rat study. Moreover, it is possible that improved myocardial function may have led to improvements in overall cardiac function had treatment extended beyond the 4\u2010week period. For this study, we chose to focus on the 20\u2010week time point, as the ZDF model is severely diabetic with compromised contractility at 20\u00a0weeks, but has not yet progressed to severe and potentially irreversible heart failure.We hypothesized that carvedilol would elicit greater improvements in cardiac function than metoprolol, but where differences in echocardiography measures of CABG patients were found they suggested that patients prescribed carvedilol in fact had poorer cardiac performance. Left ventricular internal diameters during systole and diastole were increased suggestive of dilation, and fractional shortening was decreased across patients both with and without diabetes treated with carvedilol. Patients without diabetes prescribed carvedilol also had reduced ejection fraction compared to those prescribed metoprolol, while carvedilol prescription was associated with increased HbA1c measures in patients with diabetes. However, pathological differences underlying prescription choices cannot be ascertained from the current data, and these differences are likely indicative of differential prescriptions based on clinical criteria rather than a direct effect of carvedilol. In particular, the preferential use of carvedilol in patients with left ventricular systolic dysfunction may reflect both knowledge of the COMET trial and historical restrictions on funding in New Zealand for its use outside the left ventricular systolic dysfunction. All the patients included in the study were undergoing CABG surgery, and were receiving clinically indicated \u03b2\u2010blockers and other medications, which may confound the results. However, heart tissue was not available from healthy controls, as invasive cardiac surgery is a necessary inclusion criterion, and appropriate clinical treatment must be maintained.Our findings are in contrast with a number of previous studies indicating that carvedilol has greater cardiac benefit than second generation \u03b2\u2010blockers such as metoprolol. Carvedilol decreased all\u2010cause mortality and improved ventricular remodeling following myocardial infarction compared to placebo in the CAPRICORN Trial Dargie, . SimilarWhile we observed no improvements in cardiac function with carvedilol compared to metoprolol, this does not exclude the possibility that the additional effects of carvedilol may be beneficial in the wider pathophysiology of diabetes. \u03b2\u2010blockers were thought to be contraindicated in patients with diabetes, largely related to negative metabolic side effects, but many of these are avoided by the selection of a third generation \u03b2\u2010blocker such as carvedilol Bell, . Cardiovet al. described vascular benefits of carvedilol through preservation of endothelial junctions, independent of \u03b2\u2010AR inhibition , and all patients provided informed consent. All procedures involving animals were approved by the University of Otago Animal Ethics Committee (AEC99/15) and were conducted in accordance with the New Zealand Animal Welfare Act (1999).The authors declare that they have no competing interests."}
+{"text": "R cells. Phosphoinositide-3-kinase (PI3K) and protein kinase B (AKT) were activated by sorafenib, which, in turn, increased the phosphorylation level of YB-1. In functional analyses, knockdown of YB-1 led to decreased cell migration and invasion in vitro. At the molecular level, inhibition of YB-1 induced suppression of zinc-finger protein SNAI1 (Snail), twist-related protein 1 (Twist1), zinc-finger E-box-binding homeobox 1 (Zeb1), matrix metalloproteinase-2 (MMP-2) and vimentin levels, implying a role of YB-1 in the epithelial-mesenchymal transition (EMT) process in HuH-7R cells. Additionally, YB-1 contributes to morphological alterations resulting from F-actin rearrangement through Cdc42 activation. Mutation analyses revealed that phosphorylation at S102 affects the migratory and invasive potential of HuH-7R cells. Our collective findings suggest that sorafenib promotes YB-1 phosphorylation through effect from the EGFR/PI3K/AKT pathway, leading to significant enhancement of hepatocellular carcinoma (HCC) cell metastasis. Elucidation of the specific mechanisms of action of YB-1 may aid in the development of effective strategies to suppress metastasis and overcome resistance.Hepatocellular carcinoma is one of the most common cancer types worldwide. In cases of advanced-stage disease, sorafenib is considered the treatment of choice. However, resistance to sorafenib remains a major obstacle for effective clinical application. Based on integrated phosphoproteomic and The Cancer Genome Atlas (TCGA) data, we identified a transcription factor, Y-box binding protein-1 (YB-1), with elevated phosphorylation of Ser102 in sorafenib-resistant HuH-7 Hepatocellular carcinoma (HCC) is the most common primary liver malignancy worldwide. The incidence rate of HCC has increased over the year, resulting in 300,000 to 800,000 deaths annually ,2. CurreSorafenib (Nexavar), a multikinase inhibitor, has been identified as a potent small- molecule inhibitor of Raf kinase, vascular endothelial growth factor receptor, platelet-derived growth factor receptor, Kit receptor tyrosine kinase and Fms-like tyrosine kinase 3, and it is approved for the treatment of advanced HCC by the Food and Drug Administration. In the pivotal Sorafenib Hepatocellular Carcinoma Assessment Randomized Protocol (SHARP) study and the subsequent Asia-Pacific studies, sorafenib improved the median overall survival (OS) by 2\u20133 months in patients with advanced HCC relative to placebo control . DespitePrevious studies suggest that acquired resistance results from factors that develop during sorafenib treatment. Several potential mechanisms of sorafenib resistance have been proposed to date, including epidermal growth factor receptor (EGFR) activation, c-Jun activation, autophagy, protein kinase B (AKT) activation, hypoxic environment, dysregulation of apoptosis, cancer stem cell renewal and epithelial-mesenchymal transition (EMT) . EGFR, APreviously, we employed a quantitative phosphoproteomic approach to delineate the mechanisms underlying acquired resistance of HCC to sorafenib . By inteIn this study, we focused on the molecular mechanisms of action of YB-1 in HCC development and its potential association with sorafenib resistance. The functional significance of YB-1 phosphorylation in tumor malignancy was further investigated.R cells. The proteins showing >1.5-fold changes (including 533 phosphoproteins) were classified as significantly increased in phosphorylation in resistant HuH-7R cells  (in cm3). All animal studies followed the guidelines of the Institutional Laboratory Animal Care and Use Committee of National Taiwan University. All animal experiments and animal care were performed according to institutional guidelines at the Laboratory Animal Center of National Taiwan University College of Medicine and approved by the National Taiwan University College of Medicine and College of Public Health Institutional Animal Care and Use Committee (IACUC) .Mouse subcutaneous tumor xenografts were established as described previously . When tup values set as * p < 0.05, ** p < 0.01, or *** p < 0.001 for designation of statistical significance.Statistical analyses were performed using GraphPad Prism 7 software . Data were presented as means \u00b1 SD from three independent experiments. All tests were two-tailed, and"}
+{"text": "Thunbergia laurifolia or Rang Jued has been used as an herbal tea and in folk medicine as a detoxifying agent. Cd contamination is globally widespread and a serious public health problem. The aim of this study was to determine the endogenous antioxidant enzyme activities and malondialdehyde (MDA) production of the crude dried extract (CDE) of T. laurifolia leaves, using human embryonic kidney (HEK293) and human liver (HepG2) cells as in vitro models. Moreover, the cytotoxicity including anti-cadmium (Cd) toxicity in both cells were measured. The experimental design had 3 treatment groups with combined, pre-, and post-treatments for investigating the anti-Cd toxicity, and cell viability was determined with MTT test -2,5-diphenyltetrazolium bromide). The CDE showed low cytotoxicity and increased catalase (CAT) and glutathione peroxidase (GPx) activities with decreased malondialdehyde (MDA) levels in both cell types. It was found that the CDE protected against Cd-induced toxicity in both cell types, and a synergistic combination therapy effect was seen when CaNa2EDTA, a chelating agent, was applied. Therefore, CDE can protect against Cd-induced oxidative stress in cells, possibly due to its antioxidant properties. Moreover, using the extract or drinking the herbal tea together with chelating agent should have an efficacy advantage over using the CDE or the chelating agent singly. Lipid peroxidation products, especially malondialdehyde (MDA)  fo foS-tranperoxide  and apigperoxide . In addiThe level of MDA in this experiment is shown in T. laurifolia had only low cytotoxicity with CC50 > 100 \u00b5g/mL in mouse connective tissue (L929), baby hamster Syrian kidney (BHK(21)C13), human liver (HepG2) and human colon (Caco-2) cell lines. Moreover, [To assess the potential toxicity of CDE, a cytotoxicity assay needed to be performed, and the results are shown in oreover,  suggesteHowever, close focus on cell viability or cytotoxicity against HEK293 and HepG2 cells showed some changes in a concentration dependent manner. All living cells tend to adapt to the environment by homeostasis, reacting to perturbations in a small range, to keep their balance . Moreove2 against HEK293 and HepG2 cell lines is presented in 50 of CdCl2 was 64.09 and 75.37 \u00b5mol/L for HEK293 and HepG2 cells, respectively. That the HepG2 cells had better resistance to CdCl2 may due to the liver cells having high contents of antioxidant enzymes, such as glutathione (GHS), glutathione peroxidase (GPx) and catalase (CAT) [50 of both cells was selected for testing the anti-Cd toxicity property of CDE. Cytotoxicity of the CdClse (CAT) ,52 that Effects of the CDE treatment against Cd toxicity are shown in Prior results ,46 confi2EDTA was used as a chelating agent and as a positive control  and the main function of it is binding Cd ions to form complex ring-like structures or chelates, and removal of Cd toxic metal from the desired site in the body [2EDTA before and after Cd agent for 24 h respectively. Actually, CaNa2EDTA is the most commonly used chelating agent for heavy metal exposures, especially for lead and cadmium [2EDTA does not have a specific activity for chelating only Cd, but also for other essential minerals such as Zn, Cu, Fe, Co and Mn [2EDTA therapy have been reported, including bone marrow depression, prolonged bleeding time, convulsions, respiratory arrest, and renal failures [2+ content [2+ from CaNa2EDTA dissociation may lead to Ca2+ toxicity, reducing the viability of HEK293 cell line. This is similar to the finding in [In this study, CaNa control . The res control A. Howeve control B,C, whenthe body . This ch cadmium ,69. Howeo and Mn  involvedfailures . Furtherfailures ,73. In t content . The excnding in  suggesti2EDTA in a combined-treatment was the most effective therapy. Therefore, the best concentration of CaNa2EDTA for each cell type in combined-treatment (100 \u00b5g/mL) was selected for the next experiments, studying combined therapy with the CDE against Cd toxicity. The results indicate that using CaNa2EDTA was used to treat the cell lines before, and after treatment was with the CDE. The most effective concentration of CaNa2EDTA for each cell type in the previous part was 100 \u03bcg/mL for HEK293 and HepG2, and this was used as positive control. The results showed that cell viability significantly increased when the concentration of CDE increased, compared to negative control (Cd at CC50) and to positive control (Cd at CC50 mixed with CaNa2EDTA at 100 \u00b5g/mL). This indicates that the bioactive compounds in CDE can help and support CaNa2EDTA against Cd toxin via antioxidant properties of phenolic and flavonoid compounds. Moreover, the results show that cell viability of HepG2 was higher when CDE was used at 500\u20131000 \u03bcg/mL in the pre-treatment (T. laurifolia leaves can be used as an herbal tea that can protect against Cd-induces oxidative stress. However, further study is necessary to clarify the T. laurifolia extract effects in an animal model, before clinical tests in human subjects.To study the interaction between the CDE and chelating agent, the experiment included 2 groups; pre- and post-treatments. Cd agent mixed with CaNareatment A and at reatment B comparereatment , and CDET. laurifolia contained protein, fat, ash, fiber, carbohydrate and some minerals; particularly K, P, and Mg. The present findings demonstrated that the aqueous extract of T. larifolia leaves significantly increased antioxidant enzyme activities in HEK293 kidney and HepG2 liver cells and significantly decreased MDA levels, in vitro. The extract could protect cells against Cd-induced toxicity for both cell types tested. The CaNa2EDTA agent can help cell survival better in a combined treatment than alone. Moreover, anti-Cd toxicity was higher when CDE and CaNa2EDTA were used together. Therefore, in vivo testing would be warranted before clinical tests in human subjects.Dried leaves of"}
+{"text": "Moreover, there has been insufficient evidence currently to analyze the influence of endometrial thickness (EMT) on obstetric complications and perinatal outcomes. Thus, we performed this meta-analysis to evaluate the effect of EMT on pregnancy, maternal, and perinatal outcomes in an enlarged sample size.Thin endometrium on ovulation triggering day is associated with impaired pregnancy outcomes in women after The databases Pubmed, Embase, Cochrane Libraries, and Web of Science were searched for English articles evaluating the correlation between EMT and pregnancy, maternal, or perinatal outcomes in women who underwent IVF/ICSI. We included studies that depicted a clear definition of outcomes and EMT grouping on ovulation triggering day. The EMT effect was analyzed in fresh cycle. Qualities of studies were assessed by the Newcastle-Ottawa Scale (NOS). Odds ratios (ORs) and weighted mean difference (WMD) with 95% confidence intervals (CIs) were calculated for analyzing dichotomous and continuous outcomes respectively, under a fixed or random effect model.A total of 22 pieces of literature were included for the final meta-analysis. A decreased trend towards pregnancy outcomes was observed, such as live birth rate (LBR), clinical pregnancy rate (CPR), and implantation rate (IR) in the thin endometrium groups (EMT <7 mm). In contrast, thick endometrium (EMT >14 mm) had no effect on pregnancy outcomes compared to medium EMT groups (EMT 7\u201314 mm). Moreover, thin endometrium (EMT <7.5 mm) enhanced the incidence of hypertensive disorders of pregnancy (HDP) and small-for-gestational-age (SGA) infants, and decreased the birthweight (BW) of babies.Our studies indicated that thin endometrium not only had detrimental effect on pregnancy outcomes, but also increased the risk of HDP in women and SGA of babies, or decreased BW of babies. The thick endometrium does not have an adverse effect on IVF outcomes. Therefore, patients need to be informed on possible obstetric complications and perinatal outcomes caused by thin endometrium and are encouraged to actively cooperate with perinatal care.https://www.crd.york.ac.uk/PROSPERO/display_record.php?RecordID=242637), identifier CRD42021242637.( Multiplertility . Herein,ertility . It has ertility .Many studies found that patients with thin endometrium had lower chances to be pregnant, both in fresh cycles and frozen-thawed embryo transfer (FET) cycles , 6. HoweFurthermore, maternal perinatal complications and neonatal health are of great concern following ART as well , 10. NotWe performed this review according to the Preferred Reporting Items for Systematic Review and Meta-analysis (PRISMA) statement and a registered protocol (PROSPERO registration number: CRD42021242637).in vitro fertilization) OR (intracytoplasmic sperm injection) OR ] AND [ OR  OR  OR ] AND [(live birth rate) OR (pregnancy outcomes) OR  OR  OR (obstetric outcomes) OR (treatment outcomes)]  if the consensus could not be reached. The following data were collected: authors, published year, type of study, time period, country, the number of live birth and clinical pregnancy, the number of implantation and miscarriage, the occurrence of obstetric complications , the incidence of perinatal outcomes , the definition of outcomes, sample size of thin endometrial groups and thick endometrial groups, and other related information.After excluding duplicates, titles and abstracts were screened by two independent reviewers (ZL and LC). Studies relevant to our topic were assessed for eligibility. The flow chart of search strategy is shown in Infertility women who underwent fresh cycles of IVF/ICSI treatment were included. Studies were included if these depicted the EMT of those women on ovulation triggering day and divided women into groups according to EMT. EMT, maximal distance from the endometrium\u2013myometrium junction to the outer interfaces of the endometrium in the midsagittal plane of uterus, was measured by ultrasound examination. Moreover, the outcomes in those studies should be related to pregnancy, maternal or neonatal outcomes, such as live birth rate (LBR), CPR, SGA, and so on. The definition of outcomes should be specific.We excluded studies, in patients with uterine pathology, such as fibroids, polyps, adenomyosis, and so on. Besides, donor oocytes, as a confounding factor, may affect generalizing the result as well, so studies with donor oocytes treatments were excluded. Those studies with no definition of outcomes or no EMT groups were also excluded.The primary outcome was LBR, which was defined as at least one live born baby was delivered per cycles, irrespective of the duration of pregnancy . In addiMaternal outcomes were PA  and PP . Moreover, HDP were also analyzed. Herein, HDP included gestational hypertension , preeclampsia  and eclampsia  , 14.The neonatal outcomes of dichotomous variables included SGA , large-for-gestational-age , preterm delivery  , 13. Thevia NOS independently. Disagreements between the two reviewers were settled by discussion and the third reviewer checked the accuracy of evaluation through view full-manuscript of those studies.The Newcastle-Ottawa Scale (NOS) that comprises eight items was employed to assess the quality of studies. It was used for evaluating the bias from selection, comparability and the outcomes assessment. One or two stars were awarded to each item and studies that met all criteria of the NOS would receive a maximum of nine stars. In the first item of NOS, representative cohorts were regarded as not restricted by diagnosis or by a type of ovarian stimulation protocol. Those cohort samples in fresh cycles that only received NC were also considered as unrepresentative. Moreover, studies adjusted for confounding factors  by multivariable analysis or baseline data comparison were given a star or two stars in \u201ccomparability\u201d item. Among them, age was the most important confounder that need to control. Besides, in \u201cselection\u201d and \u201coutcome\u201d items, information bias, such as the precision of measuring the EMT and evaluating the outcomes, were also taken into consideration. High-quality studies were considered as more than or equal 7 stars. Studies with medium quality had an total NOS score \u22655, but <7. Low quality studies had NOS score <5. Good quality and medium quality articles were included in the meta-analysis . Two inv2 statistics were used to evaluate the heterogeneity of studies. P <0.10 indicated the presence of heterogeneity, and I2 <50% indicated that the heterogeneity was acceptable, thus, a fixed\u2010effects model was used; otherwise, a random-effect model was used. Results were expressed as forest plots. Sensitivity analysis was conducted to examine heterogeneity and the robustness of the results. For meta-analysis of more than 10 articles, we also analyzed publication bias, which was assessed by funnel plot asymmetry and Egger\u2019s test (P <0.05 considered as significant). When the publication bias existed, trim-fill adjustment method was used to assess the effect of this bias on outcomes. Statistics tests were calculated by the Review Manager software (version 5.3). Egger\u2019s test and trim-fill analysis were analyzed by R (version 4.0.3).Given that most studies were retrospective cohort studies, we used odds ratios (OR) with 95%CI to measure dichotomous outcomes. Weighted mean difference (WMD) with 95%CI was used to analyze the association between EMT and BW. Results were combined for meta-analysis using Mantel\u2013Haenszel fixed or random effects models which depended on heterogeneity. Q statistic and IThere were 2,351 potential records by searching electronic database. After removing duplicates and screening titles and abstracts, 121 full-text articles related to our topic were retrieved for review. Of these, 82 records were excluded due to many reasons that are shown in Characteristics of included studies and patients are summarized in All women underwent the fresh cycles of IVF/ICSI treatment. Women were divided into three groups depending on EMT when analyzed pregnancy outcomes . The effect of thin endometrium on obstetric complications and perinatal outcomes was evaluated in endometrial cut-off value of < 7.5cm versus >7.5cm. The total number of reported patients and cycles that were related to LBR was about 27,225 and 31,763 respectively. The number of patients enrolled for maternal and perinatal outcomes was 4,021. The mean of female age was approximately between 29 and 36 years. COH, such as GnRH-agonist long or short protocols and GnRH-antagonist protocols, were used in patients. On hCG triggering day, the mean of E2 level of these patients was about from 1,329.78 pg/ml to 3,489.62 pg/ml.The quality of studies based on NOS is shown in 2 = 62%). Hence, sensitivity analysis was conducted to detect the stability of result by removing each study and re-analyzing the remaining studies, which did not change the direction of the effect. When one study (2 declined from 62 to 0%). Women with thick endometrium (EMT >14 mm), had no significant higher LBR than those with medium EMT (7\u201314 mm) in fresh cycles  (Women with thin endometrium (EMT <7 mm) had a significantly lower LBR compared to those women with EMT >7 mm in fresh cycles  (ne study  was excl2 = 40%) and in NC group. Since the analysis in COH stimulation group included more than 10 studies, funnel plot was presented (Twelve studies that reported CPR of women with thin endometrium in fresh cycles are shown in 2 = 64%), so we performed sensitivity analysis. When two  and the result changed . This analysis indicated that the result was not robust to some extent. Similarly, there were 10 studies in retrospective studies subgroup, so publication bias also estimated via funnel plot and Egger\u2019s test  (2 = 74%) existed  and MR  in women with thin endometrium than those with EMT >7 mm in fresh cycles  , which also corroborated our results of meta-analysis. Furthermore, a study from Nishihara et\u00a0al. also showed that CPR was significantly decreased in women with thin endometrium in fresh cycles of CC-based stimulation (P <0.05).2 = 0%) and placenta abruption . Incidence of hypertensive disorders of pregnancy was increased in women with thin endometrium, but there was no significant difference .With respect to obstetric outcomes, as shown in 2 = 0%) and babies had significantly lower BW from women with thin endometrium . No significant difference was observed in the incidence of LGA  and PTD  neonates in the thin endometrium group.Besides, perinatal outcomes, such as small-for-gestational-age, large-for-gestational-age, and preterm delivery are shown in In this review, we analyzed the effect of EMT on pregnancy, maternal, and perinatal outcomes in women after fresh cycles of IVF/ICSI. Because there was no consensus on the definition of thin or thick endometrium, we selected cutoffs of thin or thick endometrium reported in most studies for our meta-analysis, such as 7 and 14 mm in fresh cycles. Similarly, as the number of studies related to maternal and perinatal outcomes was not enough and the cutoffs of thin endometrium in these studies also have not reached an agreement, 7.5 mm that reported in most studies was selected for analyzing.We found that LBR, CPR, and IR were lower in patients with thin endometrium, which were consistence with previous studies , 22, 38.In terms of thick endometrium, there was no significant association between increased EMT and LBR, CPR, IR and MR. It should be noted that no significant difference was demonstrated between CPR in thick EMT and medium EMT group due to the substantial heterogeneity that existed among the studies. From the above results, it is clear that thick endometrium does not increase MR nor decrease CPR. Thus, thick endometrium does not have adverse effects on IVF outcomes, which is also supported by previous studies , 34, 43.Apart from pregnancy outcomes, the obstetric complications (like HDP) and the perinatal outcomes (such as BW and SGA), were revealed to be influenced by EMT. Of these, the thickness of the endometrium has a negative relationship with the incidence of HDP or SGA and a positive correlation with BW, which were in accordance with previous studies , 14, 44.Our study provided evidence that thin endometrium not only dampened the pregnancy outcomes following in IVF/ICSI, but also suppressed the fetal development, namely, increased the risk of SGA and decreased the BW of the fetus. The incidence of HDP arose, suggesting thin endometrium might also contribute to abnormal placental functions. However, because of the small number of included studies, the conclusion needs to be drawn with caution. In general, clinicians need to inform patients of possible obstetric complications caused by thin endometrium after IVF/ICSI and encourage patients to actively cooperate with prenatal examinations and receive more perinatal care after conceiving.Previous studies showed that thick endometrium had negative effect on IVF/ICSI pregnancy . Our resThis was, to the best of our knowledge, the first meta-analysis that not only explored the role of thick endometrium on pregnancy outcomes but also analyzed the effect of EMT on obstetric complications and perinatal outcomes after IVF/ICSI. Understanding these influences may enable evidence-based support to be provided.There are also some limitations in this study. Firstly, substantial heterogeneity among studies existed in some analysis, such as when analyzing the effect of thin endometrium on LBR or CPR, and the influence of thick endometrium on CPR or IR. Secondly, many of the included studies were retrospective studies and this study type is relevant to an inevitable risk of bias. Thirdly, as the different sonographers and equipment cause, the measurements of EMT are inherent with inter- and intra-variability, which might also bring some bias. Additionally, the definition of thin endometrium has not reached an agreement . In our In conclusion, our study indicated that thin endometrium had an adverse effect on LBR, CPR, IR and BW of infants, and increased the incidence of HDP in women and SGA of babies. However, it had little effect on MR, PA, and PP of patients, or on LGA and PTD among infants. More observational studies with large sample sizes and long-term follow-up or more randomized trials with preset protocols need to investigate the significance of the EMT on maternal or perinatal outcomes following in IVF/ICSI. The thick endometrium made no significant difference to pregnancy outcomes in fresh cycles.The original contributions presented in the study are included in the article/ZL and CL contributed to the design of study. ZL, LC, and LS performed studies search and data collection. ZL and CL drafted the manuscript, which was revised by KQ, CS, and HZ. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "Serpula lacrymans, we used phenotyping and integrative analysis of phylogenomic and transcriptomic data to compare this species to wild relatives in the Serpulaceae with a range of specialist to generalist decay strategies. Our results indicate specialist species have rewired regulatory networks active during wood decay towards decreased reliance on enzymatic machinery, and therefore nitrogen-intensive decay components. This shift was likely accompanied with adaptation to a narrow tree line habitat and switch to a pioneer decomposer strategy, both requiring rapid colonization of a nitrogen-limited substrate. Among substrate specialists with narrow niches, we also found evidence for pathways facilitating reversal to generalism, highlighting how evolution may move along different axes of niche space.Ecological niche breadth and the mechanisms facilitating its evolution are fundamental to understanding adaptation to changing environments, persistence of generalist and specialist lineages and the formation of new species. Woody substrates are structurally complex resources utilized by organisms with specialized decay machinery. Wood-decaying fungi represent ideal model systems to study evolution of niche breadth, as they vary greatly in their host range and preferred decay stage of the substrate. In order to dissect the genetic basis for niche specialization in the invasive brown rot fungus  Adaptation to exploit a habitat can lead species to develop specialized phenotypes. Species vary greatly in the degree of specialization to environmental conditions, nutritional resources, and competitors they experience. Species specialized in inhabiting a narrow niche may face lower competition pressure , and theRhodonia placenta includes a short window of high oxalate production [Fungi have adapted to colonize diverse habitats and their compact genome sizes make them ideal model systems to study the evolution of niche breadth in Eukaryotes on genome-wide scale. Many species are dependent on plants for their survival, either via biotrophic interactions  or in the decay of dead plant material. Wood decomposition is dominated largely by Agaricomycete fungi  and consoduction  which maoduction . Howeveroduction , 20.Saprotrophic fungi offer tractable systems to allow the assessment of the additive effect of physiological and ecological factors in characterizing niche breadth and the mechanisms underlying species separation . Fungal S. lacrymans var. lacrymans, commonly known as dry rot, causes timber decay in houses. It is a niche specialist characterized by thick mycelial cords, relatively rapid primary decay habit with a strong substrate preference for spruce and low antagonistic ability [BR fungi in the Serpulaceae  span a range of niche widths from substrate specialists with a narrow geographic range to a cosmopolitan generalist.  ability . It has  ability . Two sep ability , 28. Whi ability , 28.S. lacrymans: S. lacrymans var. lacrymans and S. lacrymans var. shastensis [shastensis is found on large logs of Abies magnifica close to the treeline in the Cascade mountain range, but is not found in the built environment to our knowledge [S. lacrymans var. shastensis provides an ideal comparator to var. lacrymans as it shares many of the niche characteristics, including a strong in vitro substrate preference for spruce and poor combative ability [S. himantioides is more commonly associated with natural environments and less frequently found in the built environment. This species is globally distributed and has a broad substrate range, associated with both hardwoods and softwoods [S. himantioides provides a more genetically diverse comparator and occupies a distinctive niche compared to its relatives, i.e., a generalist behavior with intermediate decay rates for a wider range of substrates [There are two variants of astensis , 30. Thenowledge . S. lacr ability , yet theoftwoods . S. himabstrates , 32, 33.bstrates .Picea abies (spruce), Pinus sylvestris (pine), and sucrose-based media (Czapek Dox) to establish the niche breadth of S. lacrymans variants lacrymans and shastensis, and S. himantioides and study the evolution of their decay machineries within this context. In order to survey variation within var. lacrymans from different invasive populations with contrasting levels of diversity we also de novo sequenced the genome of a Japanese strain to distinguish the European and Japanese var. lacrymans. Our analyses aimed to determine whether evidence supported a shift between generalist and specialist wood decay among the widely distributed S. himantioides that inhabits a broad host range, and the ancestor of vars. lacrymans and shastensis which are associated with narrow treeline ranges. Comparison of decay machinery with S. himantioides indicated an increased reliance on CMF and decreased reliance on PCW-degrading enzymes (PCWDEs) in S. lacrymans variants. However, decomposition ability and gene expression analysis on spruce and pine suggest a genetic basis for an underlying variability in decay between individuals of var. lacrymans and a possible route to reversal to generalism, but without the trade-off in decomposition rates seen in S. himantioides.In this study, we used a combination of experiments, phylogenomic reconstruction, and expression profiling on three different substrates, S. lacrymans var. lacrymans), SL198 (var. lacrymans), SHA17-1 (S. lacrymans var. shastensis), and MUCL38935 (S. himantioides) were maintained on Malt extract agar in the dark at 20\u2009\u00b0C. Growth experiment data were taken from Balasundaram et al. [lacrymans strains SL198 and S\u03c72 tests. Both experimental procedures are described in detail in the\u00a0P. sylvestris and spruce\u2014P. abies) in five replicates per treatment per strain. Wood shavings from untreated wood planks  were autoclaved twice at 121\u2009\u00b0C for 30\u2009min, leaving at least 24\u2009h between treatments and stored at \u221220\u2009\u00b0C until further use. The shavings were soaked in diH2O and autoclaved for a third time before ~2\u2009g of wet weight wood shavings were spread evenly across the inoculated Petri dishes. Five fungal inocula of 5\u2009\u00d7\u20095\u2009mm were placed onto a 30\u2009\u03bcm polyamide mesh  and each plug was supplemented with 200\u2009\u00b5l of 0.001% glucose solution. All treatments were grown at 20\u2009\u00b0C in the dark for 30 days prior to harvesting.Fungal strains SL200  we used a full phylogenetic approach to determine orthology relationships and gene ages within the cluster  with the \u201cweighted01\u201d algorithm and the Fisher exact test with S. himantioides (strain MUCL38935\u2009=\u2009shim) consumed all three wood types at moderate levels, on average reducing biomass by 15% on pine, 28% on fir and 34% on spruce. In contrast, the average weight loss on spruce for var. lacrymans strains (strain SL200\u2009=\u2009lacE and strain SL198\u2009=\u2009lacJ) and var. shastensis (strain SHA17-1\u2009=\u2009shas) was significantly higher than for shim . No significant difference was found when species were grown on fir.Decomposition experiments on different wood resources  show distinct wood type\u00a0- dependent responses among the four strains examined, with 0\u201369% of biomass (dry weight) consumed after 60 days growth and clear differences in decomposition among different species and wood types Fig.\u00a0. The widSerpula strains profiled also supports niche differentiation between species. Each strain was competed against each other, a second S. lacrymans var. lacrymans European isolate  using pre-colonized wood blocks of fir . In contrast, poor competitors lacE and shas did not outcompete any strain tested. The second slac European isolate  on spruce and pine, as well as \u201ccore\u201d wood-specific transcripts that were upregulated on both spruce and pine compared to sucrose  is evolutionarily conserved and driven by overlapping gene sets in all three modules  known to be involved in the degradation of PCW material were mapped in each species into oxidative CAZymes, primary\u00a0hydrolytic CAZymes acting on cellulose, hemicellulose and pectin, and accessory CAZymes metabolizing polysaccharides into simple sugars as well as assisting in PCW breakdown Fig.\u00a0. Many ofRelatively few CAZymes were specifically induced on spruce for lacE, lacJ, and shas, between 12 and 18% of all wood-induced CAZymes, compared to a range of 47 to 53% on pine  of lacE, lacJ, and shas towards spruce Fig.\u00a0. A totalFunctional enrichment analysis of the 147 genes added to the spruce module in the LCA of lacE, lacJ, and shas Fig.\u00a0. Genes iGenes gained to the spruce module in the ancestor of lacE, lacJ, and shas were also enriched for uptake and metabolism of organic nitrogen. The strongest enrichment was for the term \u201ctransmembrane transport\u201d GO:0055085 with 18 out of 25 associated genes involved in nitrogen transport e.g. oligopeptide transporters, permeases for nucleosides, purine and allantoate, three urea active transporters, and a nitrate transporter. Several terms involved in the biosynthesis of amino acids were also enriched , mostly CAZymes involved in the degradation of xyloglucan , pectin GH28) and starch (GH13 and GH15), as well as an isocitrate lyase (EC 4.1.3.1), an enzyme linking the TCA and glyoxalate cycles. The term GO:0033215 \u201ciron assimilation by reduction and transport\u201d was also enriched, driven by an iron permease  and var. shastensis (shas) are able to decompose at significantly higher rates compared to their close relative S. himantioides (shim), but that this is substrate-dependent  spp., although var. lacrymans has also been isolated from Picea (spruce) smithiana and less frequently from Pinus (pine) wallichiana, Pinus sibirica and Cedrus deodara [S. himantioides which demonstrated a more generalist decay strategy , indicate that this strain also has superior combative abilities compared to lacE and shas . These species all share the common feature of low heartwood extractive content, in particular with respect to resin acids and pinosylvin stilbenes [The shift to increased reliance on CMF in the LCA of vars. tilbenes . Differitilbenes , 53. Pintilbenes . Howevertilbenes  and we hR. placenta [In contrast, the increased weight loss caused by lacJ on pine compared to lacE, shas and shim may be due to a superior ability to detoxify pinosylvins or other inhibitory defense chemicals by this strain Fig.\u00a0. Indeed,placenta , 57 and Serpula spp., highlighting the importance of considering multiple axes in a dynamic niche space when interpreting genomic data.Taken together, our results provide a framework for understanding the evolution of fungal decay machineries in response to substrate and habitat pressures. Our conclusions are limited by the small number of isolates used in this study, and a more fine-grained mapping of niche breadths will require additional experiments, using a broader range of strains, substrates, and competitors, chosen to reflect the different habitats that these species are found in. Nevertheless, we discovered an evolutionarily conserved shift in decay strategy that coincides with increased mass loss and a more specialized niche. Integrative comparative systems, combining growth experiments and evolutionary genomic approaches provide powerful tools to understand how eco-evolutionary feedback mechanisms shape genome evolution and increasingly complex systems. Nitrogen limitation, substrate range, and fungal defense strategy in particular emerge as likely drivers shaping the decay machineries of Supplementary MaterialDatasets S1-S13"}
+{"text": "Diabetic retinopathy (DR) is a frequent complication of diabetes and through its vision-threatening complications, i.e., macular edema and proliferative retinopathy, may lead to blindness. DR is also the leading cause of vision loss in working age adults .Recent advances in therapy, particularly intravitreal administration of anti-angiogenic agents, have opened new perspectives for vision recovery. However, the availability of treatment only for the late stages of the disease and its rate of success make it urgent to understand the early alterations of DR and their progression in order to develop timely treatments before vision loss.When looking at the initial stages of DR it is said that it is present when microaneurysms and small hemorrhages appear on ophthalmoscopic examination. On histopathological examination, the first vascular changes occur in the small vessels in the form of endothelial proliferation, pericyte damage and microaneurysms . CharactUsing the examination methods now available, it may be stated that the earliest alterations that may be detected clinically in the retina in diabetes are the breakdown of the blood\u2013retinal barrier, vessel closure and alterations in the neurosensory retinal function. These alterations can be detected before ophthalmoscopic signs of DR are visible, in preclinical retinopathy .Hyperglycemia appears to be sufficient to initiate the development of DR as revealed by the development of retinopathy in animals experimentally made hyperglycemic ,8. HowevDiabetic retinopathy has been generally considered to be a microvascular complication of diabetes limiting the diagnostic and therapeutic focus to the vascular system. However, recent evidence has been accumulating suggesting that DR involves the neuronal as well as the vascular compartments.The neurosensory retina has recently been shown to be altered very early in diabetes . TogetheIt is recognized that the duration of diabetes and the level of metabolic control determine the development of DR. However, these risk factors do not explain the great variability that characterizes the evolution and rate of progression of the retinopathy in different diabetic individuals . There aWe have identified three major phenotypes of DR progression: one, characterized by slow progression, where neurodegeneration may be the only identified alteration and the retinal changes may be only a manifestation of the systemic neuropathy; a second one, characterized by occurrence of oedema resulting from breakdown of the blood retinal barrier which may occur, even in the absence of identifiable microvascular pathology; a third one identified by increased microaneurysm turnover and the presence of active microvascular lesions . In a seIt well accepted that only a subset of individuals with diabetes who develop retinal changes is expected to progress to advanced retinopathy stages and is at risk of losing functional vision during their lifetime.A fundamental characteristic of DR is that its progression varies in different individuals with the development of vision-threatening complications occurring only in a few individuals. The activity of the disease and its progression vary from patient to patient making identification of biomarkers of progression of DR to vision-threatening complications a major need. Microaneurysm turnover calculated form fundus photography images and vessel closure measured by OCTA are major candidates to fulfil this role ,13.Ischemia, identified by retinal vessel closure, appears to be the central alteration occurring in DR and the one that determines its progression to more severe stages of the disease ,14.Ashton, who has contributed so extensively to our knowledge of DR, remarked in 1974 that \u2018we must continue to look for more fundamental scientific investigations and at the same time develop new ways of examining the diabetic retina in an effort to unravel the still unsolved mysteries of diabetic retinopathy . MetricsOur group used a noninvasive, multimodal imaging approach to examine the prevalence of different disease pathways in the initial stages of the disease. Analysis of the variables representative of vessel closure, retinal neurodegeneration, and retinal edema showed a wide range of values in each ETDRS grade, demonstrating that there are very different degrees of vessel closure, neurodegenerative changes, and edema in eyes from different individuals with the same retinopathy grade.When we examined the data for correlations between the parameters that represent vessel closure, retinal thinning, and edema, we found no association between these different alterations , confirmRecently we have followed, in a longitudinal study, eyes categorized as minimal, mild, and moderate retinopathy using seven-field ETDRS grading. Of the three different disease pathways, only ischemia (vessel closure), identified by metrics of vessel density using OCTA, showed significant progression. Metrics of neurodegeneration and edema did not show progression over the period of follow-up, indicating that these two disease pathways do not appear to be associated with increased severity of the retinopathy.One of the earliest changes associated with DR is, indeed, reduced vessel density, apparently due to a decrease in retinal blood flow in selected capillaries. The reduced capillary flow in the diabetic retina is most probably related to a decrease in the number of capillaries that carry red blood cells, instead of changes in capillary diameter, because skeletonized vessel density is the metric that better detects diabetic vessel closure. The increase in vessel closure due to the number of closed capillaries to red blood cell flow is compatible with the development of preferential channels or arteriovenous shunts which have been observed on histological and trypsin-digest preparations of diabetic retinas .A study combining regional examination of the retinal circulation, including the macula region and the retinal midperiphery, in patients with type 2 diabetes in eyes with no retinopathy and different ETDRS grades of NPDR was performed using widefield Swept-Source OCTA (SS-OCTA). The study shows that retinal vessel closure is an early finding in the macular area of the diabetic retina and that this alteration increases as the retinopathy progresses in severity involving later more peripheral regions of the retina. Furthermore, SS-OCTA metrics of retinal vessel closure, allowing measurements to be performed in the macula and in more peripheral regions of the retina, may offer an objective and easier to perform alternative to ETDRS severity grading .Regional distribution of the DR lesions in the retina may reflect risk factors and may be important in defining the stage of diabetic retinopathy ,5,16. VeIn conclusion, eyes in the initial stages of retinopathy in type 2 diabetic individuals show evidence of vessel closure and neurodegenerative changes, but these are present in different degrees in different individuals even when classified as belonging to the same ETDRS severity grade. Moreover, the metrics of these changes show a wide range of values. Definite neurodegeneration and vessel closure  can occur very early in the disease process but are not present in every patient and, when present, they do not have the same rate of progression and they appear to occur independently.Furthermore, during a 3-year period of follow-up, one- or two-step worsening of retinopathy severity were associated with the presence of vessel closure and without neurodegeneration .Vessel closure identified by OCTA shows different degrees of ischemia in individuals with the same ETDRS severity grade. This observation suggests that different patients with type 2 diabetes have different microvascular responses with some individuals being able to maintain a viable retinal circulation and showing minimal changes, whereas others respond with poor capillary recruitment and progressive vessel closure.It is this variability of the retinal microvascular response in type 2 diabetes, regarding both their initiation and progression in relatively advanced stages of NPDR that we consider most relevant indicating that some individuals show steady and progressive worsening whereas others show a variable course and evidence of reversibility of their changes. These observations offer two important messages. First, the reversibility of the vessel closure opens the door for early intervention with the possibility of stopping disease progression. Second, each patient should be followed closely, and a variety of risk factors should be considered to determine a specific risk profile for that patient.OCT-angiography metrics of retinal vessel closure, more specifically vessel density measurements based on skeletonized images, obtained in a noninvasive manner that allow repeated examinations and close follow-up, are a particularly promising candidate as a biomarker of DR severity progression and are expected to impact disease management."}
+{"text": "GJA1 gene encoding connexin43, a major protein in cardiac gap junctions, plays a crucial role in the synchronized contraction of the heart and in cardiac arrhythmia. However, little is known regarding the role of GJA1 expression in the incidence of AF in patients with OSAS. All prospectively enrolled OSAS patients underwent polysomnography, electrocardiography, a 24-h Holter test, and echocardiography. Moderate-to-severe OSAS was defined as an apnea-hypopnea index (AHI) \u2265 15. Exosomes were purified from the plasma of all OSAS patients and incubated in HL-1 cells to investigate the effect of exosomes from patients with and without AF on GJA1 expression. A total of 129 patients were recruited for this study; 26 were excluded due to an AHI < 15. Of the 103 enrolled patients, 21 had AF, and 82 did not. Multivariate analysis showed diabetes mellitus, lower sleep efficiency, lower left ventricular ejection fraction, and larger left atrial (LA) size were independent predictors of AF occurrence in OSAS patients. The area under the receiver operating characteristic curve for LA with a size \u226538.5 mm for predicting AF occurrence in OSAS patients was 0.795 ; p < 0.001). GJA1 expression in HL-1 cells incubated with exosomes from OSAS patients with AF was lower than that with exosomes from patients without AF after controlling for age and sex and was negatively correlated with the AHI and oxygen desaturation index (ODI), especially during the non-rapid eye movement period (NREM) of OSAS patients with AF . LA size was an independent predictor of AF occurrence in OSAS patients. The AHI and ODI in the NREM period of OSAS patients with AF were negatively correlated with GJA1 expression in HL-1 cells, which offers a hint that GJA1 may play a crucial role in the development of AF in patients with OSAS.Obstructive sleep apnea syndrome (OSAS) is an important risk factor for atrial fibrillation (AF).  Atrial fibrillation (AF) is one of the most common heart diseases. It is a major healthcare problem due to its increasing prevalence and associated thromboembolism, heart failure, frequent hospitalizations, and mortality ,2. ObstrGJA1 gene on chromosome 6 in humans and is expressed by atrial and ventricular cardiomyocytes, vascular smooth muscle cells, endothelial cells, monocytes, and macrophages [GJA1, and other genes affecting inflammation and fibrosis may be involved in the occurrence of AF in patients with OSAS. This study investigated the predictors of AF occurrence in patients with OSAS and the effect of exosomes from OSAS patients with and without AF on the expression of GJA1 and other inflammatory and fibrotic genes that are involved in the pathophysiology of AF in HL-1 cells, with the goal of elucidating their association with the occurrence of AF.Previous studies have shown that the genes that control inflammation, gap junctions, and atrial fibrosis are associated with the pathophysiologic mechanism of AF ,14. Connrophages . Myocardrophages . Exosomerophages , and arerophages ,19. The rophages , in ordePatients with SDB were enrolled in this study from May 2019 through December 2020. Patients with an autoimmune disease, malignancy, acute infection, or age younger than 30 years were excluded from the study. Polysomnography (PSG), electrocardiography (ECG), 24-h Holter test, echocardiographic study, and peripheral blood (PB) sampling were performed after the patients were enrolled in the study. The patients\u2019 PB samples were collected between 8:00 a.m. and 9:00 a.m., and plasma from the PB of patients was used for quantification and purification of exosomes. The patient\u2019s clinical characteristics, including age, sex, and comorbidities such as a history of AF, and PSG, ECG, Holter test, and echocardiographic findings, were analyzed. The definition of heart failure in the patient\u2019s baseline characteristics was based on diagnosis during a previous hospitalization and included heart failure with a reduced and preserved ejection fraction.All patients enrolled in this study underwent an overnight PSG study. The protocol for the PSG was described in our previous study . BrieflyThe ECG and Holter tests of the studied patients were reviewed in detail by 2 electrophysiology (EP) doctors. AF was defined as no discernible repeating P waves using a standard 12-lead ECG recording or a single-lead ECG tracing of \u226530 s, according to the 2020 European Society of Cardiology guideline .Transthoracic 2-dimensional (2D) echocardiography was performed using a Sonos 7500 , following our previous echocardiography protocol ,25. The This study was approved by the Institutional Review Board of Chang Gung Memorial Hospital (IRB number: 201801943B0) and conformed to the guidelines set forth by the Declaration of Helsinki. Written informed consent was obtained from the participants before starting the study. The data underlying this article will be shared at reasonable request to the corresponding author.g for 30 min at room temperature; the supernatant was transferred into microcentrifuge tubes and aliquoted and stored at \u221280 \u00b0C. For exosome isolation, the plasma was collected and subjected to differential centrifugation at 4 \u00b0C to remove cells and cell debris: 20 min at 500\u00d7 g (twice). The supernatant was collected and incubated with thrombin, mixed gently by flicking the tube and incubating at room temperature for 5 min, and centrifuged at 10,000 rpm for 5 min, with pellets visible at the bottom of the tube. Following this, 250 \u03bcL of supernatant was transferred into a new tube, and 63 \u03bcL of ExoQuick exosome precipitation solution was added for over 1 h, and then the supernatant was centrifuged at 1500\u00d7 g for 30 min. The supernatant was aspirated, and residual ExoQuick exosome solution was spun down by centrifugation at 1500\u00d7 g for 5 min. Finally, the pellets were resuspended in 100 \u03bcL of PBS and stored at \u221280 \u00b0C. The pellet that was considered to be the exosomes was suspended in ice-cold PBS, and the protein concentration was measured using a BCA Protein Assay Kit . Exosomes were quantified using an EXOCET exosome quantitation assay , according to the manufacturer\u2019s instructions. Briefly, a standard curve was constructed using exosome standards and test samples of purified exosomes from plasma; they were then run in duplicate with an exosome quantity extrapolated from the standard curve.Exosome isolation, purification, and quantification were conducted as previously reported ,29,30. P2 in claycomb basal medium supplemented with 10% FBS , 2 mM L-glutamine (Life Technologies), 100 uM norepinephrine (Sigma-Aldrich), and 1% penicillin-streptomycin (Life Technologies) [An HL-1 cardiac muscle cell line was purchased as pooled primary cells from SIGMA . The HL-1 cells were cultured at 37 \u00b0C in an atmosphere of 5% COologies) . The celGJA1 were purchased from Applied Biosystems . The sequences of the PCR primers are listed in GAPDH. All reactions were carried out in a 10 \u03bcL final volume containing 5 \u03bcL 2X SYBR Green qPCR Master Mix , 0.2 \u03bcL primer sets, 1 \u03bcL cDNA, and 3.6 \u03bcL nucleotide-free H2O. Real-time qRT-PCR was performed in an ABI 7500 Fast Real-Time System (Applied Biosystems), and the PCR cycling parameters were set as follows: 95 \u00b0C for 20 s, followed by 40 cycles each at 95 \u00b0C for 1 s and then 60 \u00b0C for 20 s. The expression levels of the inflammation-associated genes were normalized to the internal control GAPDH to obtain the relative threshold cycle (\u0394Ct). All reactions were run in duplicate.Isolation of HL-1 cells, RNA extraction, cDNA synthesis, and qRT-PCR were performed as previously described ,33. BrieDescriptive summaries are presented for all patients and for patient subgroups. Quantitative data are reported as percentages, mean \u00b1 standard deviation, or median (interquartile range) as an appropriate approach. The differences in categorical variables between OSAS patients with and without AF were analyzed by chi-square or Fisher\u2019s exact test, and the differences in continuous data were compared using Student\u2019s t-test or the Mann\u2013Whitney U test. A binary logistic regression model was used to analyze independent predictors of the occurrence of AF in patients with OSAS. Areas under the receiver operating characteristic curve were constructed for the sensitivity and specificity of LA size to predict the development of AF in patients with OSAS. The differences in mRNA gene expression in HL-1 cells incubated with exosomes derived from OSAS patients with and without AF using the \u0394Ct values were analyzed using the Mann\u2013Whitney U test. The folds change in mRNA gene expression in HL-1 cells incubated with exosomes derived from OSAS patients with and without AF was determined by 2-\u0394\u0394Ct calculation. After controlling for age and sex, partial correlation was used to analyze the relationship between the mRNA gene expression in HL-1 cells and the occurrence of AF. The relationship between mRNA gene expression and SDB metrics in patients with AF was analyzed using Pearson\u2019s correlation. We used SPSS version 17.0 software  for all statistical analyses.p< 0.05). In addition, patients with AF had a larger LA size and lower left ventricular ejection fraction (LVEF) . There was no difference in baseline characteristics, including sex, body mass index, waistline, AHI, desaturation index, hypertension, stroke, cognitive impairment, coronary artery disease, cancer, dysthyroidism, and other PSG and echocardiographic parameters between patients with and without AF  had AF, and 82 (79.6%) did not. A total of 25 (24.3%) of the 103 patients were males, and the average age was 56 \u00b1 10 years. thout AF .Data are expressed as mean \u00b1 standard deviation, median (interquartile range) or percentage; 2D-ECHO, 2-dimensional echocardiography; AF, atrial fibrillation; AHI, apnea-hypopnea index; AO, aorta, BMI, body mass index; CAD, coronary artery disease; DM, diabetes mellitus; HF, heart failure; HTN, hypertension; IVS, interventricular septum; LA, left atrium; LVEDD, left ventricular end-diastolic diameter; LVEF, left ventricular ejection fraction; LVESD, left ventricular end-systolic diameter; LVPW, left ventricular posterior wall; LVSV, left ventricular stroke volume; NREM, non-rapid eye movement; ODI, oxygen desaturation index; REM, rapid eye movement; SpO2, oxygen saturation.p-value < 0.1, including age, DM, sleep efficiency, LA size, and LVEF, were used for the analysis of predictors of AF occurrence. Multivariate stepwise logistic regression analysis revealed that DM (odds ratio (OR): 6.511, 95% confidence interval (CI): 1.140\u201337.194; p = 0.035), lower sleep efficiency , lower LVEF , and larger LA size  were independent predictors of AF occurrence in patients with OSAS   . Since LGJA1 and higher mRNA-level expressions of TNF-\u03b1 (both p < 0.05) than OSAS patients without AF occurrence .The HL-1 cells were incubated with exosomes derived from 80 studied patients with moderate-to-severe OSAS. Among these patients, 21 had AF, and 59 did not. There was no difference in the exosome particle number between OSAS patients with and without AF. OSAS patients with AF occurrence had lower mRNA-level expressions of currence . There wGJA1 gene expression in HL-1 cells, we analyzed the correlation between PSG metrics and GJA1 gene expression after controlling for age and sex. We found there was a negative correlation between GJA1 gene expression and AHI during TST , AHI during non-rapid eye movement (NREM) , ODI during TST  and ODI during NREM  (GJA1 gene expression and AHI during rapid eye movement (REM) and ODI during REM, lowest oxygen desaturation, and the arousal index .To investigate the possible impact of OSAS on = 0.042) . There wGJA1 was lower, and TNF-\u03b1 was higher in HL-1 cells incubated with exosomes from OSAS patients with AF than in those incubated with exosomes from OSAS patients without AF. After controlling for age and sex, the gene expression of GJA1 was still lower in HL-1 cells incubated with exosomes from OSAS patients with AF. Finally, GJA1 gene expression was negatively correlated with AHI and ODI, especially during the NREM period, in OSAS patients with AF.This study had several important findings. First, OSAS patients with AF had more DM, lower sleep efficiency, a lower LVEF, and a larger LA size than OSAS patients without AF. Second, LA size was the most significant predictor of AF occurrence in OSAS patients, and the cutoff value was 38.5 mm. Third, the mRNA gene expression of Previous studies showed that DM, severity of OSAS, lower sleep efficiency, and heart failure were significantly associated with the incidence of AF in patients with OSAS ,10,24. TAccording to previous studies, LA size is not only associated with AF duration but is also an independent risk factor for AF and a predictor of recurrence of AF after therapy . With anGJA1 in HL-1 cells to become significantly lower than did exosomes from OSAS patients without AF, independent of age and sex. Further studies should be conducted to evaluate the causative mechanism of decreased GJA1 expression and AF in patients with OSAS.Cardiac fibrosis interferes with conduction by impairing intercellular coupling and leads to anisotropic conduction . InducibGJA1 gene in OSAS patients with AF, which might offer a possible underlying mechanism for AF occurrence in patients with OSAS. Moreover, a previous study showed that poor sleep quality, in terms of a shorter duration of slow-wave sleep, a factor that was independent of AHI, was associated with AF in an unselected population [GJA1 gene.AHI is the most common predictor of AF occurrence in patients with OSAS, and the use of continuous positive airway pressure is associated with a significant reduction in the recurrence of AF in patients with OSAS, irrespective of whether they undergo pulmonary vein isolation or medical treatment . Nocturnpulation . This miTNF\u03b1 gene expression in HL-1 cells incubated with exosomes from OSAS patients with and without AF disappeared after adjusting for the factors of age and sex. Further studies should be conducted to investigate whether these inflammatory mediators lead to AF directly or through comorbidities that are associated with AF occurrence. Recent studies have shown upregulation of serum HIF-1\u03b1 in patients with OSAS [Sleep disruption has been linked to increased inflammation mediators, including IL-1, IL-6, TNF\u03b1, interferon-\u03b3, and vascular endothelial growth factor in humans . These mith OSAS ,44,45. Cith OSAS . Of inteith OSAS . The disGJA1 expression in HL-1 cells incubated with exosomes from OSAS patients with AF, we did not analyze the correlation between GJA1 expression in PB from patients and that in HL-1 cells. Even though GJA1 was expressed in both atrial cardiomyocytes and macrophages, the function of the GJA1 gene is very different in both types of cells. The roles of connexin-43 in macrophages ranged from migration, antigen-presentation, and phagocytosis to polarization [GJA1 on cardiomyocytes is essential in non-immune functions in terms of maintaining myocardial electrical continuity. A previous study showed that resident macrophages may play a role in conduction abnormalities by reducing the action potential and aiding early repolarization, which may play a role in conduction abnormalities, including AF and ventricular arrhythmia [GJA1 expression in peripheral macrophages and in myocardium-resident macrophages and its role in the development of AF. Second, we did not analyze how RNAs in exosomes from patients modulated the HL-1 cells. Further studies that focus on the effect of exosomes, especially mRNAs, should be conducted to reveal their effect on GJA1 expression and AF development.This prospective cohort study had several limitations. First, although we found the rization . In contrhythmia . FurtherGJA1 genes in HL-1 cells incubated with exosomes from OSAS patients, which may play an important role in AF occurrence in patients with OSAS. GJA1 expression in HL-1 cells was negatively correlated with the AHI and ODI, especially during the NREM period in OSAS patients with AF. Further studies are needed to investigate how exosomes modulate GJA1 expression and contribute to AF occurrence in OSAS patients.This prospective cohort study of moderate-to-severe OSAS patients revealed LA size was an independent predictor of the occurrence of AF in patients with OSAS. There was less expression of"}
+{"text": "Atrial fibrillation (AF) has heterogeneous patterns of presentation concerning symptoms,duration of episodes, AF burden, and the tendency to progress towards the terminal step ofpermanent AF. AF is associated with a risk of stroke/thromboembolism traditionallyconsidered dependent on patient-level risk factors rather than AF type, AF burden, orother characterizations. However, the time spent in AF appears related to an incrementalrisk of stroke, as suggested by the higher risk of stroke in patients with clinical AF vs.subclinical episodes and in patients with non-paroxysmal AF vs. paroxysmal AF. In patientswith device-detected atrial tachyarrhythmias, AF burden is a dynamic process withpotential transitions from a lower to a higher maximum daily arrhythmia burden, thusjustifying monitoring its temporal evolution. In clinical terms, the appearance of thefirst episode of AF, the characterization of the arrhythmia in a specific AF type, theprogression of AF, and the response to rhythm control therapies, as well as the clinicaloutcomes, are all conditioned by underlying heart disease, risk factors, andcomorbidities. Improved understanding is needed on how to monitor and modulate the effectof factors that condition AF susceptibility and modulate AF-associated outcomes. Theincreasing use of wearables and apps in practice and clinical research may be useful topredict and quantify AF burden and assess AF susceptibility at the individual patientlevel. This may help us reveal why AF stops and starts again, or why AF episodes, orburden, cluster. Additionally, whether the distribution of burden is associated withvariations in the propensity to thrombosis or other clinical adverse events. Combining theimproved methods for data analysis, clinical and translational science could be the basisfor the early identification of the subset of patients at risk of progressing to a longerduration/higher burden of AF and the associated adverse outcomes.  This article is part of the Spotlight Issue on Atrial Fibrillation.Atrial fibrillation (AF) is an arrhythmia with a heterogeneous pattern of presentation, interms of symptoms, duration of episodes, time spent in AF, evolution over time, anddependence on underlying heart disease, risk factors, and comorbidities. Clinical managementof AF could markedly benefit from translational research and should provide importantfeedback on how to direct basic research to a \u2018bench to bedside and back again\u2019 process. Inthis review, we will summarize some updated data on AF susceptibility and characterization,considering AF burden and its dynamic changes, including relation to clinical factors andindices that may condition AF onset and burden, as well as patients\u2019 outcome(s). Wespecifically focused on a series of clinical factors and comorbidities that are related toAF susceptibility in terms of AF incidence, variable AF burden, and tendency for progressionto permanent AF.2DS2-VASc score is a simple stroke riskstratification tool targeted to identify patients at low risk of stroke/TE,,,per se and need to beintegrated with clinical elements to provide more targeted care. Many researchers have triedto investigate which parameters could allow an improvement in the prediction of stroke/TE ontop of the CHA2DS2-VASc score, by including assessments related to thespecific type of AF as an indirect estimate of the time spent in AF, or by measuring theburden of AF as a consequence of rhythm monitoring.,,,,,AF is associated with a substantial risk of mortality and morbidity from stroke andthromboembolism (TE),et al.The simplest approach to this topic was based on evaluating the risk of stroke inparoxysmal vs. non-paroxysmal AF. Observational studies exploring real-world practice showedthat non-paroxysmal AF types are common and predominant in daily practice.A systematic review and meta-analysis of 12 studies on nearly 100 000 patients evaluatingthe risk of stroke/TE in paroxysmal vs. non-paroxysmal AF patients, indicated that the riskof stroke is significantly higher in non-paroxysmal AF when compared with paroxysmal AF.However, the heterogeneity in study design, treatments and ascertainment of outcomes imposescaution in the interpretation of results.Since patients with non-paroxysmal AF spend more time in AF compared to those with other AFtypes, it can be hypothesized that the overall temporal burden of AF is related to anincreased risk of stroke. However, it is uncertain whether the AF pattern is truly anindependent predictor of stroke beyond the effect of the many potential unmeasuredconfounders, rather than a reflection of a different patient profile, in terms of riskfactors and comorbidities. Moreover, AF has a dynamic nature and variable evolutions,including the possibility to be progressive, with reported rates of progression to permanentAF in up to 25% of paroxysmal AF patients, depending on patient age, presentation atbaseline, underlying heart disease, and treatments/interventions.,,,,2DS2-VASc score.2 orCHA2DS2-VASc are taken into consideration.,,et al.,,Figure\u00a01).Given the limitations to quantify AF on the basis of clinical parameters and intermittentmonitoring, additional insights come from studies on patients implanted with cardiacimplantable electrical devices (CIEDs). CIEDs with an atrial lead allow a continuousmonitoring of the atrial rhythm, coupled with the storage of data on atrial tachyarrhythmiasand AF episodes presence, duration, time of occurrence, and temporal distribution, thusresulting in a precise quantification of the burden of atrial arrhythmias. For episodes ofatrial tachyarrhythmias detected by CIED, referred to as atrial high-rate episodes (AHREs)or subclinical AF, the diagnostic accuracy is highly reliable when episodes \u22655\u2009min induration are considered.,Figures\u00a0\u20022).Indices of AF susceptibility based on the AF burden pattern and dynamic changes that couldbe considered for improved patient characterization in the 4S-AF scheme are shown inTable\u00a01.There is a need for improving the characterization of AF in individual patients, asrecently suggested with the 4S-AF Scheme.Atrial cardiomyopathy and atrial structural remodelling constitute the background forincident AF and for its progression to more sustained forms. Given the variety ofunderlying structural changes and the dynamic nature of structural atrial remodelling, aspecific definition of atrial cardiomyopathy is challenging. In 2016, a consensusdocument from the European Heart Rhythm Association (EHRA)/Heart Rhythm Society(HRS)/Asia Pacific Heart Rhythm Society (APHRS)/SOLAECE proposed a pathophysiologicalclassification focused on histological changes, recognizing four classes according tothe leading mechanism of structural remodelling .Since an atrial biopsy is not routinely available, the most reliable indexes of atrialremodelling in clinical practice are atrial size and function. The role of left atrial(LA) diameter (LAD) in predicting incident AF was recognized in both the Framinghampopulation, where the risk of incident AF raised by 30% for each 5\u2009mm increase in LAD,2, independently of clinical risk factors.The predictive power of atrial remodelling is even greater when considering LA functionand specifically, LA emptying fraction (LAEF) as firstly reported in a prospective studyon sinus rhythm patients.In recent years, great developments on clinical imagery, such as LA strain-derivedquantification of LA function, could fill the gap between biological knowledge andclinical care of the arrhythmia. The role of LA strain in AF is promising since there isincreasing evidence that LA strain could be used as a valid predictor of AF occurrenceand recurrence,Higher AF burden and LA remodelling are known to be closely intertwined.Atrial enlargement was originally considered as a significant predictor of stroke andrecurrences,,Atrial cardiomyopathy encompasses a broad variety of underlying conditions andhistological abnormalities, often poorly understood. Structural atrial remodellingcertainly plays a central role in promoting AF onset and maintenance and itsrecognition, mainly in the early subclinical phase, may promote additional preventionstrategies and individualized management. However, the relationship between AF andatrial cardiomyopathy appears to be bidirectional and further studies are needed tounderstand the dynamics and the determinants of LA structural changes, also with regardto the implications for therapeutical management. The relationship between thecumulative AF burden and atrial function is not well established and requires furtherinvestigation. Both animal and human studies have found that cardioversion of AFepisodes of brief duration (<1\u2009h) either is not associated with a depression ofatrial contractile function  or results in an atrial stunningthat resolves within a few minutesFigure\u00a03,Table\u00a02S1\u2013S18and Table\u00a03S19\u2013S42  class I to class IIsymptoms to 50% in HF with NYHA class IV symptoms.,2,CHA2DS2-VASc, ATRIA) for TE risk assessment in AF and carries anincreasing yearly incidence of stroke by 0.054% for each percentage point of ejectionfraction decrease (95% CI 0.013\u20130.096%).Either HF and AF are related to adverse patient outcomes, in terms of hospitalizationrates, and increased overall mortality. Co-existing HF and AF lead to even pooreroutcomes and increased mortality than either condition alone, as well as increased riskof stroke and TE.The frequent coexistence of HF and AF raises the question of which comes first, andwhich pathways lead from a disease to the development of the other (\u2018AF begets HF andvice versa\u2019).,Hypertension is one of the most common cardiovascular disorders, currently affecting upto 50% of the global adult population.Among recently diagnosed AF patients included in the RecordAF cohort, hypertensioncarried a 1.5-fold increased risk of AF progression after a 1-year follow-up , and accounted for even greater risk in the subgroup selected for arhythm-control strategy .2,CHA2DS2VASc, ATRIA, ABC, Garfield).Hypertension is a major, independent risk factor for stroke, incremental withcoexisting AF. The close relation between BP and cerebrovascular disease/stroke riskaccounts for its relevance in common thromboembolic risk scores  is an independent risk factor for AF occurrence.Beyond being a risk factor for AF occurrence, increasing BMI has also been foundassociated with a higher burden of AF.,Several studies have investigated the relationship between obesity or being overweightand the occurrence of major adverse events in AF patients.Several mechanisms have been suggested to explain the relationship between obesity andAF. Obese patients have been found to present a number of modifications in physiologyand morphology of the heart that can directly cause occurrence of HF and AF.Diabetes mellitus (DM) is an independent risk factor for the onset of AF, as initiallyshown in the Framingham Heart Study.Patients with diabetes have a higher burden of AF since they present more frequentlywith permanent AF, as observed both in the EORP-AF registryIn real-world registries up to 20% of AF patients have DM.Several possible pathways may be involved in conditioning a milieufavouring AF onset, through an effect on action potential duration of myocytes andassociated electrical and structural remodelling.The role of hyperglycaemia associated with DM and insulin resistance, which is usuallyrelated also to hypertension and obesity, has been assessed in experimental studies.However, nowadays, the use of continuous glucose monitoring systems may offer detailedinformation on the accuracy and variability in glycaemic control in diabetic patientsand may allow us to investigate the interaction between AF and glucosefluctuations.2 had a higher prevalence of AFcompared with participants with eGFR \u226545\u2009mL/min/1.73 m2 .AF and chronic kidney disease (CKD) have a bi-directional link and the presence of CKDincreases the risk of incident AF, while the presence of AF is associated with theprogression of CKD.2.In observational studies, AF patients show a completely different pattern of AF typescomparing patients with different degrees of altered renal function, with a lowerprevalence of paroxysmal AF and a higher prevalence of permanent AF with eGFR below 50or, even greater, with eGFR below 30\u2009mL/min/1.73 m,These findings may suggest an association between AF burden and more advanced CKD, butthe interpretation of this relationship in a cause\u2013effect rapport is unclear given thepresence of other comorbidities, such as hypertension.,et al.,2 or CHA2DS2VASc seems to notimprove the predictive value of these scores.,2 and CHA2DS2VASc,it does not have an independent additive predictive value.Overall, AF patients with CKD have a significantly higher risk of adverse outcomes,including all-cause mortality, compared to those without CKD.,Renal impairment also increases the risk of bleeding. Several pathophysiologicalmechanisms have been proposed such as haemostatic defects, platelet dysfunction andaltered platelet-vessel wall-interaction etc.,Several mechanisms have been proposed to account for why AF is more common in CKDpatients.,While the prevalence of AF among coronary artery disease (CAD) populations seemsrelatively low, ranging between 0.2% and 5%, the prevalence of CAD among AF patients issignificantly high (between 13% and 46%).Even if AF during acute MI may appear to be a transient AF, long-term follow-up datashow an important rate of AF recurrence.2DS2-VASc score, defined as previous MI, peripheral arterialdisease (PAD), or complex aortic plaque.per se is associatedwith an increased risk of stroke and other cardiovascular events also in patientswithout AF.The presence of vascular disease (V) is included in theCHA,Several mechanisms have been advocated in the promotion of AF by ischaemic heartdisease. The easiest conclusion is that the presence of shared similar risk factors andthe increased prevalence with age, indeed, the concomitant occurrence of CAD and AF isonly driven by modest statistical associations. However, there are several experimentalstudies demonstrating this association. Acute atrial ischaemia creates a substrate forAF maintenance within several hours, leading to decreased conduction velocity andincreased conduction heterogeneity and shortening of atrial effective refractoryperiod.,PAD, is one of the main manifestations of systemic atherosclerosis with an increasingprevalence and incidence.There are no studies reporting specific data on the relationship between PAD anddifferential AF burden. In a study reporting data about asymptomatic PAD in AF patients,an intima-media thickness higher than 0.90\u2009mm, indicating subclinical atherosclerosis,was found more frequently associated with persistent/permanent AF, rather than withparoxysmal AF.2DS2-VASc score.,The presence of symptomatic PAD is clearly recognized as a major risk factor for strokein AF patients, also being part of CHA,The relation between PAD and AF, particularly the inverse association between ABI andincident AF, underlines a significant possible pathophysiological process. If theoccurrence of PAD is due to the presence of multiple risk factors leading toatherosclerosis, we can consider that the presence of atherosclerosis, together with thepersistence of risk factors, can perpetuate the inflammatory burden and the endothelialfunction impairment which can ultimately bring to the occurrence of AF.Despite an average prevalence of 10% among middle-aged and older subjects , up to 24 million adults in the USAremain undiagnosed.P\u2009=\u20090.003).P\u2009<\u20090.0001), and the frequency of AF was even higher (70%) inpatients with severe OSA (AHI \u226540/h). Beyond the risk of AF, OSA has been associatedwith an increased risk of stroke, but data from a US population-based case-control studyshowed that patients with OSA who experienced a stroke had a significantly higher rateof AF.The strong association between AF and obstructive sleep apnoea (OSA) has been initiallyshown by the Sleep Heart Health Study (SHHS) trial, a multicentre prospective study on6441 participants aged \u226540\u2009years.,Despite the abundance of evidence on the role of OSA in the promotion of AF, we arecurrently lacking rigorous studies on the effect of OSA on AF progression, while theavailable data seem discordant. Non-randomized studies have shown an association of OSAwith an increase in AF relapses during antiarrhythmic therapy and after electricalcardioversion or radiofrequency catheter ablation.et al.For example, Full ,,OSA and AF are, independently from each other, predictors of worse outcomes  in the general population and among severalsubgroups of patients with cardiovascular disease. The available data evidence that theclinical risk profile of AF patients with vs. without OSA is worse.Notably, the results of the Sleep Apnea Cardiovascular Endpoints (SAVE) study failed toshow positive effect of CPAP on the reduction in the composite endpoint of death fromany cardiovascular cause, MI (including silent MI), stroke, or hospitalization for HF,acute coronary syndrome (including unstable angina), or TIABeyond the number of risk factors and comorbidities shared between AF and OSA, that canexplain their association in many subjects (e.g. driven by obesity) there are manypotential mechanisms by which OSA might contribute to promote AF: (i) intermittenthypoxia, (ii) recurrent arousals, (iii) increased negative intrathoracic pressure allinducing an increased sympathetic activity, oxidative stress, endothelial dysfunction,electrical/mechanical remodelling of both atria induced by pre/post-loadmodifications.The increasing prevalence of AF with age obviously faces off against older people affectedby multiple comorbidities. Comorbid conditions can be measured using specific scores, suchas Charlson Comorbidity Index (CCI)P\u2009<\u20090.001).P\u2009<\u20090.001).P\u2009<\u20090.001 for each outcome).The relationship between CCI and AF is not fully elucidated, but since severalcomorbidities included in CCI may independently promote AF, it is logical to expect thatCCI is higher in patients with AF vs. non-AF. This association was confirmed in a largestudy analysing administrative data where a higher CCI was found in patients with AF vs.patients without AF .Beyond the mere concept of multimorbidity, the paradigm of frailty corresponds to amore complex medical syndrome, of which multimorbidity is only one dimension. Frailty ischaracterized by a reduced physiologic reserve, which makes subjects more vulnerable tostressors.Further data are still needed to fully elucidate the epidemiology and impact of frailtyin AF patients.The diagnosis of comorbidities is traditionally based on a clinical approach, andscores for comorbidities are therefore based on diagnosed diseases. Whether a clinicaldiagnosis of comorbid diseases could be replaced by a set of biomarkers (including e.g.AF burden) is yet uncertain but would enhance a more objective and quantifiableassessment of comorbid diseases and related pathophysiological alterations. The setwould include hormones, metabolomics, proteomics, genomics and epigenetics among otherdynamic markers of tissue injury.,,,,,In the future, there is a need for specific and updated pathways to employ risk factors asclinical tools for assessing AF susceptibility, in terms of risk of incident AF, and alsotargeting initiatives for AF screening.Cardiovascular Research online.Conflict of interest: GB: small speaker fee from Medtronic, Boston, BoehringerIngelheim and Bayer. GYHL: Consultant and speaker for Bayer/Janssen, BMS/Pfizer, BoehringerIngelheim, and Daiichi-Sankyo . All thedisclosures happened outside the submitted work. Other authors have no disclosures todeclare.cvab147_Supplementary_MaterialClick here for additional data file."}
+{"text": "A systematic review of the evidence should be undertaken to support the justification for undertaking a clinical trial. The aim of this study was to examine whether reports of orthodontic Randomised Clinical Trials (RCTs) cite prior systematic reviews (SR) to explain the rationale or justification of the trial. Study characteristics that predicated the citation of SR in the RCT report were also explored.Orthodontic RCTs published between 1st January 2010 to 31st December 2020 in seven orthodontic journals were identified. All titles and abstracts were screened independently by two authors. Descriptive statistics and associations were assessed for the study characteristics. Logistic regression was used to identify predicators of SR inclusion in the trial report.N\u2009=\u2009164) were cited, and 24.5% (N\u2009=\u200956) were not included but were available in the literature within 12\u00a0months of trial commencement. When a SR was not included in the introduction or no SR was available within 12\u00a0months of trial commencement, interventional studies were commonly cited. The continent of the corresponding author predicated the possibility of inclusion of a SR in the introduction .301 RCTs fulfilling the eligibility criteria were assessed. 220 SRs were available of which 74.5% (A quarter of orthodontic RCTs (24.5%) included in this study did not cite a SR in the introduction section to justify the rationale of the trial when a relevant SR was available. To reduce research waste and optimal usage of resources, researchers should identify or conduct a systematic review of the evidence to support the rationale and justification of the trial. With a wealth of trials being published in orthodontics, it is incumbent on researchers to ensure transparent reporting of interventional studies. Research waste is a known phenomenon and is a direct product of poorly conducted and reported studies as well as unnecessary duplication . SpecifiMeta-epidemiological studies aiming to ascertain the proportion of interventional trials having cited an appropriate SR have indicated that many trials still do not cite a relevant SR in their introduction as a justification for its undertaking \u20138. NeithOrthodontic RCTs published between a 10-year period (1st January 2010 to 31st December 2020) were sourced from the following seven orthodontic journals: American Journal of Orthodontics and Dentofacial Orthopaedics (AJODO), European Journal of Orthodontics (EJO), Journal of Orthodontics (JO), Angle Orthodontist (ANGLE), Orthodontics and Craniofacial Research (OCR), Journal of Orofacial Orthopedics (JOO) and Australasian Orthodontic Journal (AOJ).The phrase \u2018\u2018randomised controlled trial\u2019\u2019 was screened in the title, abstract and methodology of the article. In accordance with the Cochrane criteria for the selection of RCTs, the following inclusion criteria was used: human participants, interventions related to healthcare, experimental studies, presence of a control or comparative group, randomisation of participants to control and treatment groups, other trials with terminology in the title or abstract such as \u2018\u2018prospective\u2019\u2019, \u2018\u2018comparative\u2019\u2019, \u2018\u2018efficacy\u2019\u2019 or where an indication was given that a comparison of treatment groups was undertaken prospectively were analysed to establish whether randomisation was implemented. Studies published in English were only included. Case reports, review articles, editorials, systematic reviews and retrospective studies were excluded.https://pubmed.ncbi.nlm.nih.gov/) were searched by one author (KP) to identify eligible trials. All titles and abstracts were screened independently by 2 authors (KP and JS). Full-text articles of abstracts fulfilling the inclusion criteria were retrieved and further analysed for eligibility independently by 2 authors (KP and JS). Any disagreements in the final articles were resolved by discussion among the authors.Both journal websites and a single electronic database (Medline via PubMed: A pilot assessment of ten RCTs was undertaken between two authors (KP and JS) to ensure consistency in data extraction variables. All study characteristics were extracted by a single author (KP) and entered into a pre-piloted Microsoft Excel\u00ae  data collection sheet. A second author (JS) independently cross-checked the collected data. Any discrepancies were resolved by discussion.www.clarivate.com/webofsciencegroup/solutions/journal-citation-reports/), journal title, ethical approval , involvement of statistician , study registration (no or yes), significance of results , conflict of interest  and funding . As recommended by both the CONSORT [At the level of each RCT, the following study characteristics were extracted: year of publication, number of authors, continent of corresponding author , journal impact factor  and R Software version 4.0.3 .Descriptive statistics and associations were calculated for the inclusion of a SR in the introduction, SR not included but available in literature within 12\u00a0months of trial commencement and study characteristics. Logistic regression was used to assess associations between SR inclusion in the introduction and the study characteristics. Odd ratios, corresponding 95% CIs and p-values were calculated. Significant predictors identified during the univariate analysis were entered individually in the multivariable modell. In addition, the Boruta feature selection algorithm in R [N\u2009=\u2009164) were included in the introduction section, and 24.5% (N\u2009=\u200956) were not included but were available in the literature within 12\u00a0months of trial commencement  and SR inclusion was detected   such as the CONSORT and SPIRIT statements have strongly suggested that a systematic review (SR) is cited within the study\u2019s introduction to justify its undertaking , 4. ThisThis study showed that a higher proportion of orthodontic RCTs cite relevant SRs when compared to dental specialty journals in its entirety. The latter cohort of RCTs demonstrated that only 62.5% of RCTs have a positive SR citation . AdditioThe responsibility to reduce potentially wasteful research lies on all the stakeholders involved in its conception, design, implementation and dissemination. One specific recommendation made by Glasziou et al.  to minimoutstanding clinical uncertainty in light of the available evidence relevant for the research question and the outcome of interest at issue\u2019. Whilst it does not specifically mention the need for a SR, it does go on to mention that \u2018where no systematic review exists, applicants should make their best efforts to identify and synthesise knowledge gained in prior studies\u2019 [The EU clinical trial regulation has made strides in minimising sub-optimal research with its position statement which is due to come into effect in 2021/2022, arming local research and ethics committees alongside national competent authorities to disregard redundant research proposals. They have suggested that \u2018applicants for trial authorisation shall justify a new proposal that addresses and The methodology of the present study was based on the recommendations of the SPIRIT guidelines  which stOrthodontic RCTs published between 2010 and 2020 were only included in this study. Within this timeframe, three hundred and one RCTs were identified which represent a large enough sample to ascertain if SR are cited in the reports of RCTs. Whilst all attempts were made by the authors to ensure rigorous literature search, some studies may have been missed. This may be compounded by the fact that RCT articles were limited to English language only, hence potentially eligible RCTs may have been excluded leading to potential bias. However, through independent assessment by two authors every attempt was made to identify SRs which correlate with the primary aim of the trial and reduce potential selection bias.As per evidence-based checklists such as CONSORT and SPIRIT statements, almost a quarter (24.5%) of RCTs did not cite an appropriate SR within the introduction section as justification for the trial, when one was available. Trials where the corresponding author was based in Europe were the only predictive factor identified for positive SR citation. Further work by all research stakeholders is required within the field of orthodontics to limit research waste, ensuring finite resources are corralled for appropriately justified trials."}
+{"text": "Correction to: J Exp Clin Cancer Res 40, 140 (2021)https://doi.org/10.1186/s13046-021-01940-81Department of Gastroenterology, Putuo People\u2019s Hospital, Tongji University School of Medicine, number 1291, Jiangning road, Putuo, Shanghai, 200060, China2Department of Gastroenterology, Shanghai Tenth People\u2019s Hospital, Tongji University School of Medicine, Number 301, Middle Yanchang road, Jing\u2019an, Shanghai 200072, ChinaFollowing publication of the original article , the autThe original article has been corrected."}
+{"text": "Importantly, total uEVs were significantly higher in severe/critical patients who underwent hemodialysis (p = 0.03) and were able to predict this clinical outcome . Severe/critical patients also presented elevated urinary levels (p < 0.05) of IL-1\u03b2, IL-4, IL-6, IL-7, IL-16, IL-17A, LIF, CCL-2, CCL-3, CCL-11, CXCL-10, FGFb, M-CSF, and CTAcK. Lastly, we observed that total uEVs were associated with urinary immune mediators. In conclusion, our results show that early alterations in urinary EVs could identify patients at higher risk of developing renal dysfunction in COVID-19. This could also be relevant in different scenarios of systemic and/or infectious disease.Kidney injury is an important outcome associated with COVID-19 severity. In this regard, alterations in urinary extracellular vesicles (uEVs) could be detected in the early phases of renal injury and may be reflective of the inflammatory process. This is an observational study performed with a case series of COVID-19 hospitalized patients presenting mild-to-critical disease. Total and podocyte-derived uEVs were identified by nanoscale flow cytometry, and urinary immune mediators were assessed by a multiplex assay. We studied 36 patients, where 24 (66.7%) were considered as mild/moderate and 12 (33.3%) as severe/critical. Increased levels of total uEVs were observed ( Kidney injury can be observed in up to 40% of hospitalized patients with coronavirus disease-19 (COVID-19), where about 20% of them require renal replacement therapy ,4. SeverExtracellular vesicles (EVs) are nanosized biologically active cell fragments released to the extracellular milieu under stress conditions, which may reflect an early phase of cellular injury ,10. UrinPrevious studies have already demonstrated the importance of evaluating immune mediators in urine samples of patients with renal disease. In this regard, increased urinary excretion of cytokines such as interleukin (IL)-6, tumor necrosis factor-\u03b1 (TNF-\u03b1), monocyte chemoattractant protein-1 (MCP-1), interferon-\u03b3 10-induced protein (IP-10), and IL-10 were reported in patients with sepsis , acute iIn this study, our aim was to assess the levels of uEVs and urinary immune mediators  in hospitalized COVID-19 patients in parallel with classical laboratory parameters. Moreover, we investigated possible associations between these biomarkers and COVID-19 severity, including the need for hemodialysis as a clinical outcome. Lastly, we investigated possible associations between uEV release and urinary immune mediators.We performed a pilot study which included adult patients diagnosed with COVID-19 admitted at the Hospital Universitario Antonio Pedro  from May to September 2020. Exclusion criteria was unavailability of urine samples and patients with previous diagnosis of stage 4/5 chronic kidney disease (CKD) or prostate cancer.Demographic, clinical, and laboratory data were obtained by analyzing patients\u2019 medical records. For further analysis, we stratified patients into groups according to the clinical presentation upon admission based on the National Institutes of Health (NIH) classification: patients with mild/moderate disease  and severe/critical disease  .For the healthy control (HC) group, we used urine from healthy volunteers with no history of arterial hypertension, diabetes, kidney disease and recent infection at the time of sample collection.g for 10 min at room temperature), urine supernatant was obtained and stored at \u221280 \u00b0C. Samples were only thawed once, at the moment of analysis.Urine samples were obtained from the Clinical Pathology Service (SPC-HUAP) during the first week of hospitalization after performing routine tests required by the medical team. After an initial centrifugation , respectively. All tests were performed according to the manufacturer\u2019s instructions.g for 20 min at 4 \u00b0C. The pellet was resuspended in Annexin V Binding buffer  with Anti-Annexin-V FITC  and Anti-human Podoplanin APC . After a second centrifugation and resuspension, samples were acquired using the CytoFLEX S flow cytometer . The identification of uEVs was based on size, using calibration beads , and antibody positivity. Further analysis was performed using the software FlowJo version 9/10 . Importantly, uEV levels were normalized to urinary creatinine of all samples for the correction for any differences in urine volume ; (ii) cytokines ; and (iii) growth factors . Reading was performed using the MAGPIX Luminex instrument , and results were expressed in pg/mL.Data were expressed as mean \u00b1 SD or n (%). Differences between two independent groups were assessed by T-student or one-tailed Mann\u2013Whitney tests according to the variable\u2019s distribution. For more than two groups, we used the Kruskal\u2013Wallis test with Dunn\u2019s post-test. Differences between frequencies were analyzed with Chi-square test.We performed the receiver operating characteristic curve (ROC) for urinary measurements in order to investigate their ability to predict clinical outcomes. The cut-offs were determined from the ROC analysis according to Youden\u2019s index, which maximized both sensitivity and specificity [maximum = (sensitivity + specificity) \u2212 1] . Moreovep-values were considered significant when <0.05. Statistical analyses were conducted using the Prism software  and R.3.2.2 (R Core Team).To investigate potential confounding factors on urinary parameters, we performed a simple linear regression using total uEVs as the dependent variable and age/diabetes/hypertension as the predictor variables . Lastly,From April to August 2020, we received respiratory tract samples of 381 hospitalized patients to elucidate COVID-19 cases. Of the 126 positive patients, we were able to obtain urine samples from 41 patients. A total of 5 patients were excluded due to history of prostate cancer (n = 4) or stage 4 CKD (n = 1). Thus, for the analysis of uEVs and immune mediators, we included 36 patients.Overall, we observed a mean age (\u00b1SD) of 56.3 \u00b1 18.5 years and 20 patients were female (55.6%). According to the NIH criteria for COVID-19 severity, we identified that 24 (66.7%) patients had a mild/moderate clinical presentation and 12 (33.3%) presented a severe/critical disease at hospital admission. Demographic, laboratory, and clinical characteristics are presented in p = 0.04), in addition to requiring intensive care (p = 0.01) and hemodialysis more often (p = 0.02). Routine laboratory assessment at admission showed that these patients presented significantly higher levels of urea (p = 0.03), proteinuria , and albuminuria-to-creatinine ratio . Lastly, the frequency of death by COVID-19 was also higher in the same group (p = 0.004).As expected, we observed that patients with severe/critical COVID-19 were more frequently diabetic (p = 0.001). Podocyte-uEV counts were also higher in the COVID-19 group, but significant differences were not observed . When evaluating total uEVs according to COVID-19 severity, we observed that severe/critical patients presented higher counts , but with no statistical significance. Importantly, we also identified that patients who needed hemodialysis during disease progression presented significantly elevated levels of total uEVs at hospital admission when compared to the other severe/critical COVID-19 patients (p = 0.02). In regards to uEV analysis, we identified that COVID-19 patients presented significantly higher levels of total uEVs when compared to healthy controls (p = 0.73), diabetes (p = 0.88), and hypertension (p = 0.56) on total uEV levels  were under the lower limit of detection. Thus, for further analysis, we included 34 immune mediators. Overall, as shown in Lastly, we identified that urinary levels of immune mediators were significantly associated with total uEV count. Strong correlations (r \u2265 0.8) were observed between total uEVs and proinflammatory cytokines such as TNF-\u03b1, IL-1\u03b1, IL-1\u03b2, IL-16, and IL-17A and chemokines and other biomarkers such as CXCL-2, TRAIL, and HGF. These results are demonstrated in It is known that severe COVID-19 is a systemic disease. Specifically on the kidneys, SARS-CoV-2 can promote direct effects, but other factors can also lead to AKI in COVID-19 patients, such as ischemia, systemic inflammation, and endothelial dysfunction ,7. Thus,Our results showed that COVID-19 patients presented significantly higher levels of total uEVs in samples collected during the first days of hospitalization. This was accompanied by elevated serum urea, proteinuria, and albuminuria, especially in patients with severe/critical disease. In this context, urinary abnormalities such as proteinuria, hematuria, glycosuria, and urine pH modifications were reported in SARS-CoV-2 infection ,27. HowePodocyte-derived EVs have been investigated in different scenarios of glomerular injury, with alterations reported in obesity , diabetiImportantly, we identified that only total uEV levels were able to predict hemodialysis as a clinical outcome in severe/critical COVID-19 patients after the analysis of ROC curves. As mentioned above, patients with severe COVID-19 can develop AKI and require hemodialysis ; thus, sIn the present study, we also observed that urinary levels of cytokines, chemokines, and growth factors were increased in COVID-19 patients, especially in those with severe/critical disease. Lamb et al. (2020) previously demonstrated that COVID-19 patients presented elevated levels of proinflammatory mediators such as IL-6, IL-8, and CXCL-10 in urine . MoreoveRecently, our research group demonstrated that circulating levels of several immune mediators are markedly increased in COVID-19 patients who developed AKI independently of death as the final outcome . Here, sLastly, our results demonstrated that total uEVs are significantly correlated with urinary immune mediators in COVID-19 patients. EVs are involved in several physiological and pathological pathways, including the inflammatory process. This is evidenced by previous studies showing associations between cytokines and EVs . In addi+/phosphatidylserine (PS)+ EVs, which can bind to CD8+ lymphocytes, were associated with disease severity [In COVID-19, besides the association with the proinflammatory state, EVs were proposed as important mediators in renal thrombosis . Indeed,severity . Interesseverity . AltogetOur study has some limitations. Besides the small cohort, our main obstacle was to obtain urine samples. At the first phase of the pandemic, due to the rapid evolution of severe COVID-19, some patients died within a few hours or days after obtaining the diagnosis. Our work, at the time of conception, was not designed to explore histopathological findings and kidney biopsies in our institution were not largely indicated by assistant physicians for biosafety and epidemiological reasons. Additionally, physicians usually did not require routine urine tests and samples were not available. This also made it difficult to perform the analysis of uEVs and urinary immune mediators on a daily or weekly basis to better understand the kinetics of these biomarkers. Further studies are necessary to better characterize the subpopulations of uEVs in COVID-19 compared to other etiologies of kidney disease.Our results show that significant alterations in urinary EVs and immune mediators can be observed early in COVID-19 and are associated with disease severity. Moreover, total uEVs and immune mediators are significantly correlated with one other. This suggests that, besides the direct effects of SARS-CoV-2 on the kidneys, EV release could also be promoted by immune activation as a consequence of the multisystemic inflammation. Thus, alterations in these biomarkers could identify patients at higher risk of developing renal dysfunction."}
+{"text": "BackgroundTooth loss is a major dental health concern that has adverse consequences on the remaining dentition and on the patient\u2019s general well-being. This present study aimed to assess predictors and causes of permanent tooth extraction among students.MethodsThis national cross-sectional study in Saudi Arabia included a random sample of school students\u00a0of both genders from grades 10 to 12 (15-18 years of age) and spanned the period of September 2012 to January 2016. Demographic, social, and medical history were recorded. Moreover, a list of possible reasons for tooth extraction was discussed with participants and their parents. The questionnaire was divided into two parts. They first asked for the patient's gender, age, marital status, education level, history of smoking, and the time of the last dental visit. Periodontal and dental examinations were performed. Multivariable logistic regression was used to determine predictors of tooth loss among the sample.ResultsA total of 2,435 school students were included in the study. Notably, 24% of the students had extractions of at least one permanent tooth. Nearly 27% of female students had a permanent tooth extraction compared with only 21.7% of male students, which was statistically significant. Students who visited dentists regularly had significantly more tooth extractions (39%) than students who did not (20.6%). Multivariate logistic regression analysis showed that the significant predictors for permanent tooth extraction were age, regular dental visits, and mean probing depth (PD). Caries (15%) followed by orthodontic treatment (6%) were the main reason for permanent tooth extraction among the sample.ConclusionCaries was responsible for most of the tooth loss among the study population. Significant predictors for permanent tooth extraction were age, regular dental visits, and mean probing depth. It follows that there is a need for intensified oral health education and awareness programs in the population with an emphasis on the prevention of dental caries. Tooth loss is a major dental health concern that has adverse consequences on the remaining dentition and on the patient\u2019s general well-being .\u00a0It coulThe present study aimed to assess the prevalence, predictors, and reasons for permanent tooth extraction among school students and to synthesize data on the association between various predictors and tooth extraction in order to predict the future outcome of tooth loss based on the relation to the studied variables.Study designThis study was a descriptive cross-sectional study performed to assess the predictors and reasons for permanent tooth extraction among school students in Saudi Arabia.Ethical considerationsThe study was approved by the Research Ethics Committee of the Faculty of Dentistry, King Abdulaziz University (073-09-12).Inclusion and exclusion criteriaThe study included and aimed at sampling a randomly selected group of healthy school students and a group of school students with medical conditions that did not have a correlation with periodontitis. The students were from grades 10 to 12 (15-18 years old) and were of both genders.\u00a0Students of parents who refused to sign the consent form or who rejected the periodontal examination and students with medical conditions (conditions reported to have a relation to periodontitis)\u00a0were excluded from the study. No students were admitted to the study without their parent's approval.Research toolA random sample of school students from grades 10 to 12 (15-18 years of age) of both genders was included.\u00a0The study spanned from September 2012 to January 2016. A \u201cMultistage Clustered Sampling Design\u201d was employed and was reported in detail in an earlier report .\u00a0Subject0 = No plaque1 = Thin film of plaque at the gingival margin, visible only when scraped with an explorer2 = Moderate amount of plaque along the gingival margin; interdental space free of plaque; plaque visible with the naked eye3 = Heavy plaque accumulation at the gingival margin; interdental space filled with plaqueThe severity of gingival inflammation was assessed using the Loe & Silness Gingival Index according to the following criteria:0 = Normal gingiva, no inflammation, no discoloration, no bleeding1 = Mild inflammation, slight color change, mild alteration of gingival surface, no bleeding2 = Moderate inflammation, erythema, swelling, bleeding on probing or when pressure applied3 = Severe inflammation, severe erythema and swelling, tendency toward spontaneous hemorrhage, some ulcerationStatistical methodologyThis study was analyzed using IBM SPSS\u2122 22.0.0 . This is a simple descriptive statistic used to define the characteristics of the study sample variables through the form of counts and percentages for categorical and nominal variables, while mean and standard deviation are used to present the continuous variables. To test the bivariate relationship between tooth loss and categorical and continuous variables, this study used the chi-square and independent t-test, respectively. These tests were done with the assumption of normal distribution. Multivariable logistic regression was used to determine predictors of tooth loss among the study sample. A model with backward conditional elimination with the enter criterion=0.05 and elimination=0.10 was utilized. Statistical significance was set at P <.05.The total number of students in the selected cities were\u00a0Riyadh n=713), Jeddah (n=657), Dammam (n=506), Abha (n=297), and Tabuk (n=262). Table 3, JeddahTable Table Tooth loss can provide information regarding the availability of dental care, the prevalence of dental disease, and attitudes toward tooth loss. In the present study, 24% of the students had extractions of at least one permanent tooth. It was then found that 40.9%\u00a0in a cross-sectional study carried out in the Eastern Province of Saudi Arabia on the same age group also had such extractions ,16.Despite numerous previous studies having examined the nature of tooth loss and dental extractions of permanent teeth in adults, surprisingly little information exists regarding the loss of permanent teeth among students . One musThe present study shows a tendency for more extraction of permanent teeth among females than males. This is consistent with some earlier studies\u00a0-21. OtheRegarding the extraction of permanent teeth, several conclusions were obvious from the data. First, caries was the most common reason for extraction. These results are consistent with a study conducted in the US, which reviewed 2000 records and concluded that 53% of tooth extraction was mainly due to caries and pulpal pathosis . AnotherIn the present study, tooth extraction due to periodontitis was found in only 1% of the sample. This finding is consistent with a study on an Italian sample that reported that no one under 20 years of age had extractions due to periodontal diseases . These fThe prevalence of malalignment (mainly crowding) and the need for orthodontic treatment among the Saudi population were reported to be high .\u00a0TherefoAs limitations to the current study that can be included in future studies, patient compliance should be studied in association with the reason for tooth extraction. In addition, detailed periodontal bacterial pathogenic screening tests, including a Porphyromonas gingivalis species count, as well as saliva buffering capacity and production tests can be explored in order to test the degree of association with the reasons for tooth extraction.The present study indicated that caries is the principal reason for tooth extraction among the studied population of school children in Saudi Arabia, followed by extraction for orthodontic reasons. Therefore, prevention of caries in the population must begin early in life to identify those at risk. Certainly, this study and many others support the need for further studies defining these risk factors and exploring methods of intervention for those at risk."}
+{"text": "In this study, the air quality index (AQI) of Indian cities of different tiers is predicted by using the vanilla recurrent neural network (RNN). AQI is used to measure the air quality of any region which is calculated on the basis of the concentration of ground-level ozone, particle pollution, carbon monoxide, and sulphur dioxide in air. Thus, the present air quality of an area is dependent on current weather conditions, vehicle traffic in that area, or anything that increases air pollution. Also, the current air quality is dependent on the climate conditions and industrialization in that area. Thus, the AQI is history-dependent. To capture this dependency, the memory property of fractional derivatives is exploited in this algorithm and the fractional gradient descent algorithm involving Caputo's derivative has been used in the backpropagation algorithm for training of the RNN. Due to the availability of a large amount of data and high computation support, deep neural networks are capable of giving state-of-the-art results in the time series prediction. But, in this study, the basic vanilla RNN has been chosen to check the effectiveness of fractional derivatives. The AQI and gases affecting AQI prediction results for different cities show that the proposed algorithm leads to higher accuracy. It has been observed that the results of the vanilla RNN with fractional derivatives are comparable to long short-term memory (LSTM). These pollutants are the residual gases and particles emitted from vehicles, industries, and due to climate change , that gives the following:\u03b1 order Caputo's fractional derivative. Caputo gave this definition of fractional derivative in 1967 for overcoming the limitations of RL derivative [\u03b1 > 0, n \u2212 1 \u2264 \u03b1 < n for a constant c is zero. This version increased the applicability of fractional derivatives in modelling real world problems. Thus, in this study, Caputo's version of fractional derivatives has been used.It is called the rivative . The Cap(iv)Gr\u00fcnwald\u2013Letnikov (GL) fractional derivative: The limit definition of fractional derivative was given by Anton Karl Gr\u00fcnwald and Aleksey Vasilievich Letnikov in 1867 and 1868, respectively [\u03b1 > 0, the \u03b1-order GL derivative of function f is expressed as follows:ectively . Withouth is the step size and Here, Factional calculus is a 300-year-old branch of mathematics that deals with derivatives and integrals of noninteger order, i.e., order can be any number, be complex or real. Earlier, this domain was only theoretical involving rigorous calculations, but these derivatives are used in practical applications as well , 30. SevClearly, the limit definition of first-order derivative shows that evaluation of derivative involves usage of only two points. But it can be seen from limit definition of fractional derivatives and from the Caputo's definition in equation  and equa\u03b7 and \u03b1 are the learning rate and fractional order of differentiation, respectively. Chen [The nonlocality of fractional derivatives has been the major motivation for their application in different domains. Fractional calculus has been successfully used for air quality prediction \u201341. Fracly. Chen  employedly. Chen , recurrely. Chen , convoluly. Chen , and evely. Chen .i is the output neuron, \u03a6(ui(s)) and xi(s) are the actual output and the expected output of the ith neuron at time s, and ui(s) = \u2211j\u2208\u03a9wijvj(s) at time s, where wij(s) is the weight of a signal from jth neuron to ith and vj(s) is the output of jth neuron at time s; then, the update rule becomes as follows:\u03b7 is the learning rate and \u2207wij\u03b1 represents the factional gradient w.r.t wij. Now, \u2207wij\u03b1E(s) can be evaluated by applying the approximated chain to the error function. The actual chain rule applicable on fractional derivatives is complicated and involves special mathematical functions; thus, several approximated chain rules have been developed for fractional derivatives. [f then for a small h,h\u27f60, we geth\u27f60\u0394\u03b1f(x)/h\u03b1=f\u03b1)((x),d\u03b1f(x) \u2248 \u0393(1 + \u03b1)df(x)0 < \u03b1 < 1. Using this result,In this section, we introduce the fractional-order truncated backpropagation through the time algorithm on the RNN with 10 neurons in a layer. This backpropagation algorithm considers the truncated depth of the input data and the state of the network, which makes the algorithm computationally efficient. For the implementation of the backpropagation algorithm, the mean squared error at an instant is considered as follows:vatives. \u201353 The cAfter using the abovementioned fractional chain rule, we get\u2202E(s)/\u2202uj(s)=\u03b4j(s), then0CDx\u03b1xp = \u0393(p + 1)xp\u2212\u03b1/\u0393(p \u2212 \u03b1 + 1), for p > \u22121, then the following holds:Suppose quations , 16), a, a\u2202E(s)/ng holds:\u2202\u03b1uis\u2202wjiIn this study, the vanilla RNN has been employed to predict the AQI value of a day based on the previous sequential AQI data of five different cities. RNNs are capable of learning the sequential pattern of historical data. Furthermore, the accuracy of the system has been improved by incorporating memory into the system using the fractional gradient descent algorithm.In this study, the continuous AQI data have been used to predict future unseen AQI values. For each city, a continuous-time patch of around 1000 data points has been used from the AQI dataset. The sample data can be seen in th) day. Training and testing sets have 600\u2013800 and 100 data values, respectively. For the initializing of weights for the models, Xavier's initialization has been used with 0.1 learning rate; 80 epochs were used to train the model for each city and each fractional order. The vanilla RNN has been used in the proposed model which has a single-layered architecture with 10 nodes in it. The fractional gradient-based RNN model is built from scratch using the NumPy library, and the Pandas library is used for data preprocessing. Forward propagation and fractional gradient-based back propagation as given by  have beeviz root mean squared error (RMSE) and mean absolute percentage error (MAPE) have been employed to assess the performance of the proposed model in the prediction of AQI of different Indian cities and the major pollutants in one of those cities. The lesser the value of RMSE and MAPE, the better the performance of the predictor. These errors measure the performance of forecasting, climatology, and regression analysis for verifying the experimental results. The detailed information related to these parameters is provided.The standard evaluation metrics for forecasting models ot is the expected output for iteration t, which are observed for N times.The root mean square error (RMSE) is the square root of the average of the squared difference between the actual and predicted value. RMSE can be expressed by the following expression:ot is the expected output for iteration t, which are observed for N times. This is a form of percentage error, which has helped in the analysis of the proposed model in different situations.The mean absolute percentage error (MAPE) is the average percentage of the absolute difference between the actual and predicted values divided by the actual value for each time period . MAPE caThis section describes the AQI dataset of five cities chosen, the results obtained by using the proposed approach on the AQI data, and the discussion of comparison between predictions of LSTM and the proposed approach on different fractional orders. The performance of networks is measured using RMSE and MAPE.http://cpcb.nic.in/). The dataset consists of daily air quality levels at various stations across multiple cities in India which are obtained by averaging out the hourly value of AQI. Indian cities chosen for the analysis includes Kolkata, Hyderabad, Bengaluru, Patna, and Talcher. Basic information related to these cities is as follows:Kolkata (22\u00b034\u203203\u2033N 88\u00b043\u203257\u2033 E), located in West Bengal, is a tier I and the seventh most populous city of India with third-most populous metropolitan area. The concentration of pollutants such as sulphur dioxide and nitrogen dioxide remains within the limit, but the presence of particulate matter in air is high and is increasing over the years. Due to this, air pollution is severe and is causing respiratory ailments such as lung cancer.Bengaluru (12\u00b058\u203244\u2033 N 77\u00b035\u203230\u2033 E), located in Karnataka, is also a tier I and the third most populous city of India with fifth most populous metropolitan area. Bengaluru is also considered as \u201cSilicon Valley of India\u201d because it is the nation's top IT exporter. This IT hub region is the most polluted and is causing several environmental issues. Due to the large population, Bengaluru generates tonnes of solid waste which is polluting the environment. Thus, the large population and IT hub of Bengaluru is the major reason for air pollution.Hyderabad (17\u00b021\u203242\u2033 N 78\u00b028\u203229\u2033 E), located in Telangana, is also a tier I and the fourth most populous city of India with sixth most populous metropolitan area. Again, due to the large population, increased economic activity, and rapid urbanization, tonnes of solid waste are generated, and disposal of such waste becomes hazardous and pollutes the environment. The particulate matter (PM10) dispersed in the atmosphere causes around 2500 deaths each year.Patna (25\u00b036\u20320\u2033 N 85\u00b06\u20320\u2033 E), located in Bihar, is a tier II city with a high population. Air pollution is a major issue in this city. The situation in winter becomes even worse due to dense smog, leading to an increase in mortality. Patna was declared as the second most air polluted city in India, in the WHO survey of 2014.Talcher (20\u00b057\u20320\u2033 N 85\u00b013\u203248\u2033 E), located in Angul district of Orissa, is a tier III city. This is a small city with less population, but Talcher has the country's biggest coalfield with the highest coal reserve of around 52 billion tonnes. The presence of these coal mines leads to air pollution.The AQI dataset of five cities for 2015\u20132020 is considered which is publicly available at the official portal of the Central Pollution Control Board, Government of India  and genetic algorithms can be employed for optimizing the order of differentiation. Fractional gradient descent can be used with suitable architectures for different cities. Through the results of predictions of various gases of the city, we can find a better way to develop in a sustainable way."}
+{"text": "Fap-knockout mice bearing CRCLM allografts. Mechanistically, bevacizumab treatment induced hypoxia to upregulate the expression of fibroblast growth factor\u2013binding protein 1 (FGFBP1) in tumor cells. Gain- or loss-of-function experiments revealed that the bevacizumab-resistant tumor cell\u2013derived FGFBP1 induced FAP\u03b1 expression by enhancing the paracrine FGF2/FGFR1/ERK1/-2/EGR1 signaling pathway in HSCs. FAP\u03b1 promoted CXCL5 secretion in HSCs, which activated CXCR2 to promote the epithelial-mesenchymal transition of tumor cells and the recruitment of myeloid-derived suppressor cells. These findings were further validated in tumor tissues derived from patients with CRCLM. Targeting FAP\u03b1+ HSCs effectively disrupted the co-opted sinusoidal blood vessels and overcame bevacizumab resistance. Our study highlights the role of FAP\u03b1+ HSCs in vessel co-option and provides an effective strategy to overcome the vessel co-option\u2013mediated bevacizumab resistance.Vessel co-option has been demonstrated to mediate colorectal cancer liver metastasis (CRCLM) resistance to antiangiogenic therapy. The current mechanisms underlying vessel co-option have mainly focused on \u201chijacker\u201d tumor cells, whereas the function of the \u201chijackee\u201d sinusoidal blood vessels has not been explored. Here, we found that the occurrence of vessel co-option in bevacizumab-resistant CRCLM xenografts was associated with increased expression of fibroblast activation protein \u03b1 (FAP\u03b1) in the co-opted hepatic stellate cells (HSCs), which was dramatically attenuated in HSC-specific conditional   Colorectal cancer (CRC) is the third most common and the second most fatal malignancy in the world . SpecifiHepatic stellate cells (HSCs), also known as perisinusoidal cells, are a group of contractile and secretory cells closely attached to the periphery of hepatic sinusoid endothelial cells . HSCs ar+ HSCs with the FAP\u03b1-activated prodrug Z-GP-DAVLBH (Fibroblast activation protein \u03b1 (FAP\u03b1) is a type II integral membrane serine protease that can specifically cleave N-terminal benzyloxy carbonyl\u2013blocked (Z-blocked) Gly-Pro (Z-GP) dipeptide-linked substrates . FAP\u03b1 isP-DAVLBH  effectivhttps://doi.org/10.1172/JCI157399DS1). The HGPs in CRCLM xenografts were then examined, and our results showed that HGPs of HCT116 CRCLM xenografts in the vehicle group were mainly DHGP and PHGP, while RHGP was the main form in tumors from the bevacizumab-resistant group  or HSC-specific conditional Fap\u2013knockout mice (\u0394GfapFap) to generate the intrinsically bevacizumab\u2013resistant CRCLM allografts  and the negative control cells (LX-2Vector) were generated   . In addi16 cells . These e16 cells . Taken tHomosapiens proteome database, 17 upregulated proteins were identified in bevacizumab-resistant tumors  to generate HT-29shNC cells or FGFBP1-knockdown HT-29shFGFBP1 cells, respectively  , 38. We nd FGFRs , A and Bnografts , indicatnografts , and EGRpromoter , we propP1 cells . The abo-2 cells , A and B29 cells . Moreove29 cells . FurtherP1 cells . ChIP-qPP1 cells .FGFBP1 or HT-29shNC CRCLM xenografts  or FAP\u03b1-overexpressing plasmid   . ELISA sor cells . As a re16 cells , decreas16 cells . Howeveror cells . Moreove16 cells , increasR1Vector . These f+ HSCs compared with Chemo treatment  and HSCs has been demonstrated to exert critical regulation in maintaining the liver\u2019s physiological function in liver fibrosis and cirrhosis . Tumor-iCyGB gene to inhibit the transformation of HSCs into myofibroblasts and thereby alleviates liver fibrosis and cirrhosis . All cell lines were tested negative for mycoplasma using the Mycoplasma Detection Set (M&C Gene Technology). For hypoxia experiments, cells at 60% confluence were transferred into a sealed hypoxia chamber filled with 5% CO2, 1% O2 and 94% N2 at 37\u00b0C and cultured for the indicated times.Human CRC cell lines (HCT116 and HT-29) were obtained from ATCC. The HSC line LX-2 was purchased from Sure Biological Technology, and the mouse CRC cell line (MC38) was purchased from BeNa Culture Collection. These cell lines were cultured in DMEM (Gibco) with 10% FBS (ExCell Bio) and 1% penicillin-streptomycin (Gibco) at 37\u00b0C in a humidified atmosphere containing 5% COFap\u2013knockout mice were generated as described previously with some modifications (fl/flFap mice (T052266), and Gfap-Cre mice that were backcrossed with C57BL/6J mice (T004857) were obtained from GemPharmatech. \u0394GfapFap (Gfap-Fap knockout) mice were generated by crossing Gfap-Cre mice and fl/flFap mice, and littermate fl/flFap mice were used as wild-type controls. All mice were maintained in a specific pathogen\u2013free facility. The genotypes of transgenic mice were identified by PCR analysis of genomic DNA from tail snips using specific primers . HCT116 cells were transfected with pCMV3-HIF1A and empty plasmid using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer\u2019s instructions. For FGF2-, FGFR1-, EGR1-, and HIF1\u03b1-knockdown experiments, LX-2 cells were transfected with siRNA targeting FGF2, FGFR1, EGR1, or negative control, and HCT116 cells were transfected with siRNA targeting HIF1A or negative control (Genepharma). The sequences of shFGFBP1 are listed in Lentivirus containing luciferase vector GV260 (Ubi-MCS-firefly Luciferase-IRES-Puromycin), lentivirus containing either a FAP\u03b1 or FGFBP1 overexpression plasmid and the corresponding vector GV367 (Ubi-MCS-SV40-EGFP-IRES-puromycin), and lentivirus containing shFGFBP1 plasmid and the corresponding vector GV248 (hU6-MCS-Ubiquitin-EGFP-IRES-puromycin) were constructed by Genechem. Lentivirus infection was carried out according to the manufacturer\u2019s instructions. HCT116-luc cells were generated by infection with lentivirus containing luciferase vector. LX-2 cells stably overexpressing FAP\u03b1 were obtained by infection with lentivirus containing FAP\u03b1 overexpression plasmid. HCT116 cells stably overexpressing FGFBP1 were obtained by infection with lentivirus containing FGFBP1 overexpression plasmid. 7 cells/mL, and a total of 2 \u00d7 105 tumor cells in 20 \u03bcL were injected into the left main lobe of the mouse liver. Tumor-bearing mice were randomized into the vehicle and bevacizumab groups on the seventh day. The treatment group of HCT116 or HCT116-luc xenografts was injected intraperitoneally (i.p.) with 10 mg/kg bevacizumab (Roche) twice a week for 7 weeks, and HT-29 xenografts were treated with 10 mg/kg bevacizumab for 2 weeks, while the vehicle group received IgG. In HCT116 or HCT116-luc CRCLM xenografts, tumors were collected on the 14th, 28th, and 42nd day after bevacizumab treatment to determine the characteristics of acquired bevacizumab resistance. To assess tissue hypoxia status, 50 mg/kg hypoxyprobe-1  was intravenously (i.v.) injected into mice for 30 minutes before being euthanized by CO2 asphyxiation. For therapeutic administration, mice bearing acquired-bevacizumab-resistance HCT116 xenografts and intrinsically bevacizumab\u2013resistant HT-29 xenografts were treated with bevacizumab combined with i.v. injection of 2 mg/kg Z-GP-DAVLBH once every other day for 14 days, or combined with 1.8 mg/kg FGF2-neutralizing antibodies (Sigma-Aldrich) once a week for 2 weeks (i.v.), or combined with 20 mg/kg PD166866 (Selleck) once every other day for 3 weeks (i.p.). MC38 cells (2 \u00d7 105 cells/mouse) were injected into the liver of \u0394GfapFap and fl/flFap mice after administering tamoxifen (100 mg/kg) via oral gavage once every other day for a total of 3 times, and the tumor-bearing mice were sacrificed by CO2 asphyxiation 2 weeks later. HCT116Vector, HCT116FGFBP1, HT-29shNC, and HT-29shFGFBP1 cells were injected into the left main liver lobe of male BALB/c nude mice. On the seventh day, HCT116FGFBP1 and HT-29shNC mice were treated with 20 mg/kg PD166866 once every other day for 3 weeks (i.p.) or 1.8 mg/kg FGF2-neutralizing antibodies once a week for 2 weeks (i.v.). Tumor tissues were collected for further histological analysis.The HT-29  or HCT11Bioluminescence imaging was conducted to monitor tumor growth in vivo using the IVIS Lumina LT imaging system (PerkinElmer). Mice bearing HCT116-luc CRCLM xenografts were injected (i.p.) with 150 mg/kg D-luciferin (Yeasen) before isoflurane (RWD Life Science) anesthesia. Live animal imaging was acquired 10 minutes after the injection of D-luciferin. For the quantification of total radiance efficiency, a region of interest was drawn around the tumor and radiance efficiency was measured.a), DHGP (b), and PHGP (c) in the liver-tumor interface of each image were calculated and converted into micrometers. The percentage RHGP was then quantified according to the following formula: a/(a + b + c) \u00d7 100. The same calculation method was used for the quantification of DHGP and PHGP. For immunohistochemical staining, deparaffinization, rehydration, antigen retrieval, permeabilization, and blocking were performed on the sections, followed by incubation of primary antibodies overnight at 4\u00b0C. The slides were then incubated with HRP-conjugated secondary antibodies and visualized using a DAB staining kit (Servicebio). Immunofluorescence staining was visualized with iF488-Tyramide, iF555-Tyramide, or iF647-Tyramide using TSAPLus Fluorescence Kits (Servicebio). Immunofluorescence staining of LX-2 cells was performed using Alexa Fluor 488\u2013conjugated secondary antibodies (Invitrogen) and Alexa Fluor 594\u2013conjugated phalloidin (Invitrogen). Image analyses were performed using an Olympus BX53 inverted epifluorescence microscope or a Zeiss LSM 800 confocal microscope. All positive cells were counted in high-power fields at a magnification of \u00d7200, \u00d7400, or \u00d7640. Five areas from each section were randomly selected to count the percentage of positively stained cells and to calculate the mean staining extent using Image-Pro Plus 6.0 software (Media Cybernetics). Detailed information on the primary and secondary antibodies is listed in Formalin-fixed tissue samples of mouse or human CRC liver metastasis tissues were embedded in paraffin and sectioned at a thickness of 5 \u03bcm. Hematoxylin and eosin (H&E) staining was performed according to standard procedures. The types of histopathological growth patterns were evaluated using ImageJ software (NIH), and the full-length pixels of RHGP (+ (Biolegend) with purity greater than 95% by flow cytometry. LSECs were isolated using anti-CD146 beads (Miltenyi Biotec) and characterized as CD146+ (Miltenyi Biotec) with purity greater than 95% by flow cytometry as described previously . A single-cell suspension of tissues was obtained using a GentleMAC tissue processor (Miltenyi Biotec) according to the manufacturer\u2019s instructions. Tumor cells were isolated using anti-EpCAM beads (Miltenyi Biotec) and characterized as EpCAMeviously . MDSCs weviously .6 cells/mL of reagent according to the manufacturer\u2019s instructions. High-throughput full transcriptome sequencing and subsequent bioinformatics analysis were performed by Applied Protein Technology Co., Ltd. In brief, mRNA was purified from total RNA using a NEBNext Poly(A) mRNA Magnetic Isolation Module  according to the manufacturer\u2019s instructions. RNA-seq libraries were generated using ribosomal RNA\u2013depleted RNAs with the NEBNext Ultra II RNA Library Prep Kit (New England Biolabs) following the manufacturer\u2019s instructions. Libraries were controlled for quality and quantified using the Bioanalyzer 2100 system (Agilent Technologies). RNA-seq Illumina Libraries were prepared and sequenced on an Illumina GAIIx sequencer with 101 base-length read chemistry. For the data analysis, raw data in FASTQ format were processed through in-house Perl scripts. All downstream analyses were based on high-quality clean data. The index of the reference genome was built using Hisat2 v2.0.5 (http://daehwankimlab.github.io/hisat2/), and the paired-end clean reads were aligned to the reference genome using Hisat2 v2.0.5. For the quantification of gene expression level, FeatureCounts v1.5.0-p3 (https://sourceforge.net/projects/subread/files/subread-2.0.0/) was used to count the read numbers mapped to each gene. The fragments per kilobase of transcript per million mapped reads values of each gene were calculated based on the length of the gene and read count mapped to this gene. Analysis of differential expression was performed using the DESeq2 R package v1.16.1 (https://bioconductor.org/packages/release/bioc/html/DESeq2.html), and the differentially expressed mRNAs and genes were selected with statistical significance (P < 0.05) by R package edgeR. The volcano plot revealed the distributions of log2(fold change) and P values for the differentially expressed genes. The GO terms (http://www.geneontology.org) of these differentially expressed genes were annotated.Total RNA was extracted using TRIzol Reagent (Servicebio) at a ratio of 10n = 3 per group) were homogenized in SDT buffer  and quantified using the BCA protein assay reagent (Thermo Fisher Scientific). Then, 20 \u03bcg of protein per sample was resolved by SDS-PAGE and digested according to the FASP (filter-aided sample preparation) procedure (http://www.matrixscience.com) and Proteome Discoverer version 1.4 (http://www.thermoscientific.com/en/product/proteome-discoverer-software.html) and searched against the UniProtKB/Swiss-Prot Homosapiens proteome database . For protein quantitative analysis, the relevant parameters and descriptions were as follows: enzyme = trypsin, max missed cleavages = 2, fixed modification = carbamidomethyl (C), variable modifications = oxidation (M), peptide false discovery rate (FDR) \u2264 0.01. The protein ratios were calculated based on the median of only unique peptides of the protein. Proteins with fold change greater than 1.2 or less than 0.83 and P value (Student\u2019s t test) less than 0.05 were considered differentially expressed.Tandem mass tag (TMT) quantitative proteomic analysis was performed as described previously with some modifications . Bevacizrocedure . After tACTB gene using the 2\u2013\u0394\u0394Ct method. All primers are shown in Total RNAs were extracted using the E.Z.N.A. Total RNA Kit I (Omega Bio-Tek) and reverse transcribed to cDNA using the All-in-One cDNA Synthesis SuperMix (Bimake) according to the manufacturers\u2019 instructions. RT-qPCR was performed using SYBR Green I Master (Genestar) according to the manufacturer\u2019s instructions. The relative mRNA expression level of target genes was normalized to the endogenous (Ct 2% input sample \u2212 Ct IP sample), where Ct = threshold cycle of the PCR reaction.ChIP assays were conducted using the SimpleChIP Enzymatic Chromatin IP Kit according to the manufacturer\u2019s instruction . Antibodies are listed in Cells and tumor tissues were harvested and lysed with ice-cold RIPA lysis buffer (Thermo Fisher Scientific), and the Pierce BCA Protein Assay Kit was used to measure the concentration of proteins. Western blotting was performed as described previously  and the 4) were seeded on the top chamber with 100 \u03bcL serum-free medium, and the conditioned medium of LX-2 cells was added into the bottom wells. Both the invaded and migrated cells were fixed with 4% paraformaldehyde for 30 minutes, stained with crystal violet solution (Sigma-Aldrich), and imaged under the Olympus BX 53 microscope. To evaluate the migration of human MDSCs, MDSCs (1 \u00d7 105) were plated in the upper chambers and different conditioned medium was added in the lower chamber. After incubation for 6 hours, the number of MDSCs in the bottom compartments was counted as described previously (Cell invasion and migration in vitro were determined using 8 \u03bcm pore size Transwell chambers (Corning Costar) with or without Matrigel (BioCoat) diluted 3:1 using PBS. HCT116 cells (2 \u00d7 10eviously .5 cells/well) or MDSCs (1 \u00d7 105 cells/well) were seeded in black 96-well clear-bottomed plates for 24 hours prior to the experiment. The intracellular Ca2+ levels were measured using a Screen Quest Fluo-8 No Wash Calcium Assay Kit (AAT Bioquest) according to the manufacturer\u2019s instructions. Absorbance (Ex/Em: 485/535 nm) was determined at steady state. Different conditioned medium was added after the steady-state measurement to induce calcium release from intracellular sources and determine intracellular calcium levels (50 \u03bcL/well). Absorbance was monitored for 225 seconds, and several readings were obtained using an EnVision Multimode Plate Reader (PerkinElmer).HCT116 cells (1 \u00d7 10FGFBP1 or HT29shNC cell\u2013conditioned medium for 24 hours.LX-2 cells were cocultured with LSECs derived from bevacizumab-sensitive or -resistant HCT116 CRCLM xenografts using 0.4 \u03bcm pore size Transwell chambers (Corning Costar) with LSECs culture in the upper chamber. LX-2 cells were added to the lower chambers of Transwell plates and cultured overnight until they grew to 50% confluence, and the cells were then cultured with HCT116An FGFBP1 ELISA Kit (Boster) and CXCL5 ELISA Kit (Solarbio) were used to measure FGFBP1 or CXCL5 levels in the cell culture supernatant according to the manufacturers\u2019 instructions.LX-2 cells treated with vehicle or Z-GP-DAVLBH (1 \u03bcM) for 24 hours were harvested and stained with an Annexin V\u2013FITC/PI assay kit (Beyotime). The percentage of apoptotic cells was measured using a FACSCanto system (BD Biosciences).3 cells/100 \u03bcL medium. Following incubation with or without Z-GP-DAVLBH for 24 hours, the cell viability was assessed using a Cell Counting Kit-8 (Targetmol) assay according to the manufacturer\u2019s instructions.LX-2 cells were seeded in 96-well plates at 5 \u00d7 10The contrast-enhanced CT scans of CRCLM patients preoperatively treated with Chemo or Chemo+Bev were available for the analysis of morphological response to therapy using a method based on previously published morphological response criteria . MorpholFor scoring of the pathological response of CRCLM patients with RHGP treated preoperatively with Chemo or Chemo+Bev from contrast-enhanced CT scans, the largest area of the tumor surface area was measured using the greatest diameter and the greatest perpendicular distance, while the reduction rate was calculated as (tumor area prior to treatment \u2013 tumor area following treatment)/tumor area prior to treatment. Pathological response was scored independently by 3 experienced pathologists using the above method.t test, and differences between more than 2 groups were evaluated using 1-way ANOVA followed by Tukey\u2019s post hoc test. Survival curves were constructed using the Kaplan-Meier method and analyzed by the log-rank test. All statistical analyses were performed using GraphPad Prism 7.0 software. A P value of less than 0.05 was considered significant.Data are presented as mean \u00b1 SEM. Differences between 2 groups were evaluated using the 2-tailed, unpaired The human CRC liver metastasis tissue specimens and CT scan images used in this study were approved by the Clinical Ethics Committee of the First Affiliated Hospital of Jinan University, and written informed consent was received from participants prior to inclusion in the study. The animal experiments were approved by the Institute of Experimental Animal Ethics Committee of Jinan University.Proteome data are available via ProteomeXchange with identifier PXD030202. The raw RNA-seq data have been deposited in NCBI\u2019s Gene Expression Omnibus (GEO) and are accessible through the GEO Series accession numbers GSE207976, GSE208084, and GSE208091.DZ, W Ye, and MC designed the study. MQ, SF, MC, MH, YL, QM, WL, WD, and W Yin carried out the experiments. MQ, SF, MC, MH, and X Li performed data analysis. JP, CJ, WH, LX, SQ, XC, and JH provided pathology review and assessment of clinical and preclinical samples. JH, QQ, LD, X Liu, YY, and CS helped in the experimental design and manuscript writing. MC, MQ, and SF wrote the manuscript. DZ, W Ye, MH, TL, and QQ revised the manuscript. The order of the co\u2013first authors was determined on the basis of their efforts and contributions to the manuscript. All authors have seen and approved the final version of the manuscript."}
+{"text": "Dental trauma may have a severe impact on the social and psychological wellbeing of a patient. Most cases of dental injuries involve anterior teeth, especially the maxillary upper incisors. Crown fractures, with or without pulp exposure, are the most common trauma in permanent dentition. There are many methods of management, in which the initial state of the pulp, the time since the injury, and the presence of an accompanying injury play a key role. This case report aimed at showing a possible conservative treatment after complicated tooth fracture that consisted of partial pulpotomy followed by adhesive reattachment of the tooth fragment using a technique based on heated resin composite. Such a specific procedure represents a conservative approach to traumatic coronal lesions, providing a suitable opportunity to maintain the tooth vitality, aesthetics, and function. Indeed, reattachment of tooth fragment using a composite/adhesive is a simple technique to achieve excellent results in terms of aesthetic and function. Traumatic dental injuries (TDIs) concern mostly children and young adults . TDIs maThe treatment of complicated crown fractures according to the International Association of Dental Traumatology   includesDuring diagnosis, it is strongly recommended to take a parallel periapical radiograph. Additional radiographs are required if there are signs and symptoms of other potential injuries. For soft tissue injuries, X-rays of the lip and/or cheek are needed to look for tooth fragments or external debris. In the event of suspicion of other injuries, especially root fractures, crown\u2013root fractures, or lateral luxations, the clinician should consider using cone beam computerized tomography (CBCT). This examination enables the determination of the location, extent, and direction of the injury . The decWhen a tooth fragment is available, it should be reattached; if it is not available, it is recommended to cover the dentin with a glass-ionomer or a bonding agent and composite resin . If a poThe favorable outcomes include asymptomatic teeth with positive response to pulp sensibility testing, good quality restoration, and continued root development in immature teeth. Treatment outcome depends on the severity of the injury, quality, and timeliness of initial care, and recall protocol  [A 15 year old male patient (case #1) experienced a blunt trauma during basketball game. The patient suffered from a complicated fracture of the crowns of 11 and 21 A,B. The In case #2, a 21 year old male patient reported to dental office immediately after a complicated crown fracture of the crowns of 11 during a tennis game C,D. The In both cases, teeth were vital with thermal and electrical stimulus; no mobility and symptoms of other trauma were detected during clinical and X-ray examination A,B. The In case of complicated crown fractures, vital pulp therapy (VPT) interventions include direct pulp capping (DPC), partial pulpotomy (PP), and complete pulpotomy (CP) .Based on the analysis of the clinical conditions and additional tests, it was decided to perform PP with adhesive/composite reattachment in case #1, as well as in case #2, maintaining the vitality of the treated teeth. PP was chosen as the treatment method following the admission of patients within 14 days after injury, no caries, and vital and asymptomatic pulp.In general, VPT has a high success rate. However, clinical factors such as the vitality of the pulp, the time from exposure to intervention, age of the patient, other coexisting injuries, the cause of exposure, and the extent of exposure may influence the favorable outcome . AdditioThe diagnosis of injured tissue plays a key role, which can often be difficult in the case of trauma. Additionally, the tests used for sensitivity assessment , which are dependent on neural response, may not be reliable in the first days after trauma . In the It is important to consider that the success of VPT is also influenced by factors such as proper infection control (rubber dam isolation), bleeding control, selection of the capping material providing a tight seal, and a final restoration.In the presented cases, after administrating the anesthesia of 2% lidocaine with 1: 80,000 adrenalines not alkalinized , and performing a total rubber dam isolation to avoid cross-contamination, the partial pulpotomy was executed through high-speed bur under continuous saline irrigation.Various local anesthetics and different vasoconstrictors concentration are used in VPT after trauma. Based on the available literature, the active substance and its concentration demonstrated no effect on the treatment results . On the The ability to control bleeding is an important factor determining the success of treatment, as prolonged bleeding is supposed to be a sign of an irreversible inflammatory process. The time needed to stop bleeding varies from 2 to 25 min, with no effect on the prognosis of pulpotomy . MoreoveFor bleeding control, the hemostatic agents are recommended. True hemostatic agents such as ferric sulphate or hydrogen peroxide are not recommended, due to the risk of masking the inflammation of the radicular pulp . SeveralAn ideal pulpotomy material should be biocompatible, non-toxic, induce hard tissue formation, and exhibit a disinfecting properties . The matCH exhibits bactericidal properties, high pH, and, therefore, it is characterized by the ability to neutralize acids and lipopolysaccharides . These pMost CSMs, such as mineral trioxide aggregate (MTA), are based on Portland cement, consist mainly of dicalcium or tricalcium silicates, and are mixed with water ,32. TheyOne of the major aesthetic complications after pulpotomy with white and grey MTA can be the discoloration of the tooth ,37. It iThe powder of MTA is mixed with distilled water (3:1 ratio) to obtain a wet, gel-like consistency . For mixMany systematic review papers and meta-analyses report that there is no significant differences between MTA and CH regarding the survival rate of pulp, both for permanent immature teeth and teeth with closed apices ,50,51,52The calcific metamorphosis is an adverse effect of the MTA and CH application ,52. It iWhen a well-hydrated intact tooth fragment is available, which fits to the remaining crown without interfering with the patient\u2019s occlusion, the first-choice treatment should be adhesive reattachment of the fragment. It does not always have to be stored in a humid environment; a study shows that a dehydrated fragment was attached to the tooth with the use of additional retention elements, and after a 15 month follow-up, the tooth retained its vitality, functionality, and natural aesthetics . HoweverCompared to conventional restorative techniques, the reattachment of tooth fragments exhibits several advantages: the original shape, color, brightness, and texture of the enamel surface . The incMany different types of adhesive systems  and different intermediate materials e.g., paste and flowable composite materials, adhesives, or glass ionomer cements, can be applied ,63,64,65Some authors recommend pre-reattachment/initial modification of remaining fragments, e.g., dentine grooves, over-contouring, chamfering, or beveling, to expand the enamel surface, increasing adhesion and ensuring higher fracture resistance of such restorations ,67. ThesThe bond strength between adhesive systems and calcium-silicate-based materials is also an important aspect. The in vitro results indicate that the bond strength of the resin-based materials to the MTA is favorable for the total-etch technique ,71,72. OIn the present cases, no modification was carried out before the reattachment procedure. In both cases, the selective enamel etching both on the tooth and the fragment was performed using a 36% phosphoric acid gel  under rubber dam isolation. Then, a self-etching two-component adhesive system  was applied as per manufacturer\u2019s instruction, and air-dried with a strong stream of air for 5 s to completely remove the excess adhesive. This was finally light-cured for 20 s . Subsequently, a thin layer of enamel (Shade A2) mass composite  was applied directly on the tooth as an intermediate material to reattach the fragment to the tooth. The composite was heated up to 54 \u00b0C in a warming device  to increase the degree of polymerization, a better adaptation of the fragment on the tooth and to provide easier management of the excess removal . Next, pOcclusal adjustment involves the development of an acceptable central relation contact position for the patient, ensuring acceptable lateral and protrusion guidance. This is necessary to eliminate premature contacts and bad guidance, which could contribute to excessive forces concentrating within the reattached fragment, thus, causing it to detach. Therefore, the presented cases were performed using a diamond bur . Due to the perfect fit of tooth fragments and prior excess removal, minimal occlusion adjustment was needed.Polishing provides a smooth surface of the teeth, thus, reducing the accumulation of dental plaque. While it is important, polishing removes the fluorine-rich enamel layer and should, therefore, be carried out selectively .The restored teeth were first finished using fine and extra-fine diamond burs ), and finally polished using either a Soflex discs coarse, medium, fine, and super-fine grit Sof-Lex disk  in a slow-speed hand piece for 30\u2009s each.The follow-up visits after trauma injury are of paramount importance and, therefore, mandatory. The control should consist of an interview, radiological examination, and pulp sensitivity tests. It enables the early detection and implementation of appropriate treatment to avoid long-term complications. The most common post-traumatic problems include pulp infection and necrosis, pulp canal obliteration (PCO) or root resorptions.In the case of the complicated crown fractures, the recommended follow-up visits are as follows: after 14 days, 6\u20138 weeks, 3 and 6 months, and one year after the injury. In the case of the presented cases, the check-ups were carried out in accordance with the recommended scheme, and the positive results obtained after one year by interview, radiological examination, and pulp sensitivity cold/hot test indicate the success of the treatment.If the pulp is exposed, biological treatment procedures should be performed. It is of paramount importance in the case of patients with open apices, as the preservation of the vital pulp ensures further physiological root formation. The prognosis of direct pulp capping with use of CH shows a success rate of 54\u201390% ,79. In aThe reported success rate of partial pulpotomy in permanent dentition with complicated crown fractures ranges from 87.5% to 100% ,53. The The employment of adhesive techniques increases the success rate of the reattachment procedure by up to 84\u201393% ,59. CaseMoreover, the use of toothpaste containing biomimetic hydroxyapatite for home management after reconstruction can reduce discoloration and hypersensitivity more effectively than conventional fluoride toothpaste ,89."}
+{"text": "Caenorhabditis elegans and Drosophila melanogaster and plays important roles in the regulation of essential life functions in each organism. OA and TA are thought to act as the mammalian homologs of epinephrine and norepinephrine respectively, and when triggered, they act in response to the various stressors in the fight-or-flight response. 5-HT regulates a wide range of behaviors in C. elegans including egg-laying, male mating, locomotion, and pharyngeal pumping. 5-HT acts predominantly through its receptors, of which various classes have been described in both flies and worms. The adult brain of Drosophila is composed of approximately 80 serotonergic neurons, which are involved in modulation of circadian rhythm, feeding, aggression, and long-term memory formation. DA is a major monoamine neurotransmitter that mediates a variety of critical organismal functions and is essential for synaptic transmission in invertebrates as it is in mammals, in which it is also a precursor for the synthesis of adrenaline and noradrenaline. In C. elegans and Drosophila as in mammals, DA receptors play critical roles and are generally grouped into two classes, D1-like and D2-like based on their predicted coupling to downstream G proteins. Drosophila uses histamine as a neurotransmitter in photoreceptors as well as a small number of neurons in the CNS. C. elegans does not use histamine as a neurotransmitter. Here, we review the comprehensive set of known amine neurotransmitters found in invertebrates, and discuss their biological and modulatory functions using the vast literature on both Drosophila and C. elegans. We also suggest the potential interactions between aminergic neurotransmitters systems in the modulation of neurophysiological activity and behavior.Neurotransmitters are crucial for the relay of signals between neurons and their target. Monoamine neurotransmitters dopamine (DA), serotonin (5-HT), and histamine are found in both invertebrates and mammals and are known to control key physiological aspects in health and disease. Others, such as octopamine (OA) and tyramine (TA), are abundant in invertebrates. TA is expressed in both  This communication relies in part on vesicular fusion mediated by Ca synapse . Expressdulthood .Drosophila but unlikely to be synthesized in C. elegans  neurons, paired left/right and dorsal/ventral in the nose tip. Left/right neuronal pairs innervate the cuticle of the head and tail, namely anterior deirid (ADE) and posterior deirid (PDE) neurons, respectively , which is synthesized by GTP cyclohydrolase I (GTPCH), the first and rate limiting enzyme in that process , which converts L-DOPA into DA , which is unique to Drosophila and acts as a negative regulator of TH, likely in part through an interaction with VMAT and a coupling of DA synthesis to its transport . The tiosophila . VMAT isiew, see ). Indeediew, see . Also, cransport .C. elegans,  homolog that is encoded by bas-1 . Typically, it is synthesized in the cytosol (or recycled from the extracellular space) and packaged into synaptic vesicles for exocytotic release, after which it binds to post-synaptic DA receptors  for subsequent relay of the signal downstream see . It is ihetamine .C. elegans, DA is one of four biogenic amines which acts through its receptors to modulate important behaviors like locomotion in a manner similar to that seen in mammals (dop-3) and DOP-1  provides a mechanism through which extra-synaptic DA is regulated, with behavioral effects downstream through the recruitment of G\u03b1o and G\u03b1q  deletion mutants. Experiments have shown that excess DA causes worm immobilization, which can be observed through a swimming induced paralysis (SWIP) assay in which the greater the DA presence at the synapse, the faster the animals become paralyzed  studies have been done with aralyzed . Since Dterparts . DOP-2 ise of DA . In addi studies . HoweverDrosophila by Fumin , which converts DA to 3,4-dihydroxyphenylacetic acid (DOPAC)  and in by dat-1 . This acng sites . Unlike  (DOPAC) . MAOs ar (DOPAC) . MAO-B i (DOPAC) . Drosophorganism . Besidesosophila  to form C. elegans DAT is that it likely executes its function through an interaction with DOP-2 (Synaptic transmission above). Moreover, there are suggestions that the C. elegans SNARE protein, syntaxin 1A homologue, UNC-64 represses DAT , responsible for converting DA to DOPAC or to 3-methoxytyramine (3-MT), respectively. Of the proteins that bear some homology to MAO, AMX-1, AMX-2, and AMX-3 each have 35\u201340% amino acid similarity with MAO-A and MAO-B. AMX-2 has an \u03b1 helical domain similar to the C-terminal domain in MAO-A . The D2-like receptor group includes D2, D3, and D4 receptor subtypes, which upon agonist binding generally inhibit adenylyl cyclase through coupling to G\u03b1i/G\u03b1o subunits, and further inhibit cAMP production  of DA receptors, which decades ago were classified into D1-like and D2-like groups based on their physiology and mechanism of action . Both groduction . Dop2R  is classified as a D1-like receptor while DOP-2 and DOP-3 are D2-like receptors (dop-4) is a D1-like receptor that is unique to invertebrates . A case in point is the mediation of synaptic vesicle fusion by DOP-2 and the presynaptic acid sensing Na+ ion channel (ASIC-1), encoded by asic-1. FRAP studies on asic-1 and dop-2 deletion mutants have shown that when measuring recovery rates, vesicular fusion was inhibited in asic-1 mutants, but quickly recovered in dop-2 mutants. We note that it is not clear whether the ASIC-1 effect on synaptic release is direct or indirect, but this is an important question that merits further inquiry. These results and others suggest that DOP-2 may physically associate with the inhibitory unit of G\u03b1i of GPA-14 (DA receptor function is similarly conserved in iewed in ). Three eceptors . DOP-4  is expressed on dopaminergic neurons   , althougDVMAT null mutants can be partially-rescued with DA neuron-specific expression of TH , a behavior exhibited by worms in the presence of its food, bacteria, is another activity that is modulated by DA . One keyordingly . This laDrosophila and C. elegans, the first step in 5-HT synthesis is the rate-limiting hydroxylation of the amino acid tryptophan, which generates 5-hydroxytryptophan and is catalyzed by tryptophan hydroxylase (TPH)  ; Table 1yptophan . In wormn  catalyzby bas-1 . In wormby bas-1 .Drosophila ortholog of SERT) takes up excess 5-HT from the synaptic cleft , making it an essential component of 5-HT regulation . dSERT  was identified about two\u00a0decades ago as the only C. elegans reuptake transporter , SER-4 (encoded by ser-4), SER-5 (encoded by ser-5), and SER-7 (encoded by ser-7) (mod-1), a 5-HT-gated chloride channel (mod-5) (At least four types of G-protein serotonergic receptors have been identified in y ser-7) . Additio channel , has bee channel . SER-5 i channel . Moreove channel . This ex (mod-5) .Drosophila is composed of approximately 80 serotonergic neurons, distributed in a spatially diverse pattern of clusters in the CNS  slows walking speed, whereas inhibition of the same serotonergic neurons enhances it . The re mammals . Along t mammals ). 5-HT a mammals . Moreove mammals . Further mammals . Intrigu mammals .Drosophila  and slowing when worms encounter the food . ImportaEnhanced slowing response (ESR) can be described as a complimentary behavior to basal slowing. It involves food-deprived animals  exhibiting an enhanced slowing response that allows them to spend a maximum amount of time foraging on bacteria, their food source, and it is regulated by a 5-HT neural circuit .q homolog egl-30, activates vulval muscles, whereas binding to G\u03b1o homolog goa-1 does the opposite  that transforms TA into OA   and encoin worms ; Table 1in worms ; Table 1in worms . Once syand 5-HT . In mammosophila .C. elegans begins with the conversion of the amino acid tyrosine to TA by TDC (encoded by the gene tdc-1 in worms) (tbh-1)  ; Table 1 (tbh-1) . The cat (tbh-1) . TA is a (tbh-1) . As prevA and OA .Drosophila, previously known as \u201cOctTyrR\u201d class. TAR1 shows preference for TA binding over OA . TAR2 is abundant in the brain, thoracoabdominal ganglion and in the midgut of the adult fruit fly, whereas the orthologous TAR3 is highly expressed in the adult eye and in the tubule and hindgut of larvae. The Malpighian tubules, which are the primary urine production organs, are the best characterized system for studying tyraminergic signaling in native fly tissue.One prominent feature of the tyraminergic system is its receptor classes, which include TAR1, the first type of TA receptors present in  over OA  and inhi+ levels . The TAR+ levels . A thirdmilarity , but theC. elegans, SER-2 (encoded by ser-2), TYRA-2 (encoded by tyra-2), TYRA-3 (encoded by tyra-3), and LGC-55 (encoded by lgc-55) are the tyraminergic receptors that have been discovered thus far. SER-2, TYRA-2, and TYRA-3 are GPCRs and TA appears to interact with the G\u03b1 subunit of each  was later found to regulate feeding rates through a blockage of 5-HT release. The behavior is exhibited when the worms come across inedible food , sex pheromone production (moth), appetite (blowfly), and muscular contractions (locust) .Drosophila larvae  in which head movement is inhibited and regulated by TA  to play a key role in linking the neural circuits that regulate locomotion and head movement . Upon aned by TA , throughed by TA   Figure. Both neDrosophila OA receptor, OAMB , SER-6 (encoded by ser-6), and OCTR-1 (encoded by octr-1), each coupled to G\u03b1q, G\u03b1q and G\u03b1i, respectively . All 3\u00a0eceptors . SER-3 heceptors . When OAeceptors .Drosophila. These behaviors range from locomotion (see below) to the tracking of CO2 during flight -negative subfraction of that cluster (a group of about twenty cells) serves as an aggression-triggering complex, whereas fru positive subfraction acts as the courtship-triggering center  in TA levels . Moreovq, which probably works through SER-3 in SIA neurons  is responsible for the de novo biosynthesis of histamine from L-histidine at presynaptic release sites  . It is se sites . Histamise sites   . Carcinid by tan , a gene ears ago . Becauseora triansentless (ort) and histamine-gated chloride channel subunit 1 (HisCl1) have been identified in Drosophila. The latter receptor has been shown to act in photoreceptor neurons to synchronize flies\u2019 behavioral rhythms with light-dark cycles  cause abnormal temperature preference behaviors  and are consistent with work in rodents showing that DAT forms a protein complex with D2R and that its cell surface expression levels are regulated by that interaction . It shoAn exciting potential new ground lies on the follow up to the discovery of LOVIT as a VMAT-independent mechanism for the transport of histamine. Its localization to photoreceptors indicates a narrow cell-type expression, unlike VMAT, which is found in multiple aminergic neuron-types. However, it will be interesting to know if there are other organisms or monoaminergic systems that use this transporter in a similar fashion.Drosophila, C. elegans and other invertebrates. In many of these roles, the neurotransmitters not only affect behaviors, but they also regulate the actions of other biogenic amines, sometimes in a cooperative fashion and at other times in an antagonist sense. A plethora of new knowledge has been uncovered from work in both model systems, much of it paving the way for interesting insights into the diverse roles that monoamine neurotransmitters play in the regulation of essential life processes, and in how the molecules themselves are regulated.Biogenic amines play a myriad of roles in the regulation and modulation of neuronal functions in"}
+{"text": "In the last decade, there has been remarkable progress in research toward understanding and refining the hallmarks of cancer. In this review, we propose a new hallmark - \u201cpro-survival autophagy.\u201d\u00a0The importance of pro-survival autophagy is well established in tumorigenesis, as it is related to multiple steps in cancer progression and vital for some cancers. Autophagy is a potential anti-cancer therapeutic target. For this reason, autophagy is a good candidate as a new hallmark of cancer. We describe two enabling characteristics that play a major role in enabling cells to acquire the hallmarks of cancer - \u201ctumor-promoting microenvironment and macroenvironment\u201d and \u201ccancer epigenetics, genome instability and mutation.\u201d\u00a0We also discuss the recent updates, therapeutic and prognostic implications of the eight hallmarks of cancer described by Hanahan et al. in 2011. Understanding these hallmarks and enabling characteristics is key not only to developing new ways to treat cancer efficiently but also to exploring options to overcome cancer resistance to treatment. The transformation of a normal cell into a neoplasm is a complex process. Hanahan et al. summarized hallmarks of cancer including six core hallmarks, two emerging hallmarks and two enabling characteristics . The bidNew hallmark of cancerPro-survival AutophagyAutophagy is an important process in cell death, conservation of protein homeostasis and maintenance of normal organelle function, as it removes damaged structures\u00a0under cell environment stress conditions. Autophagy can occur in three different pathways: chaperone-mediated autophagy (with the participation of intermediate ligand chaperone proteins like HSP70s), microautophagy , and macroautophagy (where cytoplasmic components are sequestered into the autophagosome and degraded on lysosomes) .Autophagy is essential for oncogenic K-Ras-induced malignant cell transformation in human breast epithelial cells, as the mRNA protein levels of ATG5 and ATG7 (autophagy-specific genes) were increased in cells overexpressing K-Ras and that targeted suppression of these genes inhibited cell growth and tumor formation . Tan andAlthough not totally clear, some studies suggest a positive correlation between autophagy and epithelial-mesenchymal transition (EMT), which is needed in cancer progression and metastasis, probably via p62 (an autophagy adaptor protein) and tumor growth factor \u03b2 (TGF-\u03b2), which is the most important regulator of EMT in human cancers . KnockdoBecause autophagy facilitates cancer cells' survival in an environment with hypoxia and metabolic stress, it reduces tumor necrosis and consequently the infiltration of macrophages in the primary tumor, that is a required step for metastasis . Also, aCancer stem cells (CSCs) that play an important role in tumor recurrence and resistance to anti-neoplastic treatment strategies, seem to have a role in tumor maintenance and function related to autophagy. Some autophagy markers like ATG5, ATG12, and LC3B were found to be overexpressed in dormant stem cell-like breast cancer cells  and BECLAutophagy, as an important survival mechanism against various cellular stresses, can induce resistance to various anti-cancer therapeutic agents  by reducing ROS damage, blocking apoptosis, and maintaining the CSC pool . Some thThe better comprehension of pro-survival autophagy leads to studies assessing classes of therapeutic anti-cancer targets in this field, most of them with preclinical evidence, but some are still ongoing clinical trials. One of them are class III isoform of phosphoinositide 3 kinase (PI3K), that induces autophagy by generating PI3-phosphate, needed for the formation of autophagosome membrane . WortmanThe importance of pro-survival autophagy is well established in tumorigenesis, as it is related with a lot of events in cancer progression and critical for some of them in various types of cancers. Besides that, autophagy is a potential anti-cancer therapeutic target. For this reason, autophagy is a good candidate for a new hallmark of cancer.Enabling characteristicsTumor Promoting Microenvironment and MacroenvironmentHanahan et al included tumor-promoting inflammation as one of the enabling characteristics of cancer . Most tuTumor initiation:\u00a0The inflammatory microenvironment increases mutations and genetic instability either by producing ROS and reactive nitrogen intermediates or via cytokines that stimulate ROS ,36.\u00a0InflTumor promotion:\u00a0Inflammatory response induces genes promoting cell proliferation and survival .\u00a0Tumor aTumor metastasis and invasion:\u00a0Inflammatory myeloid cells and cancer cells produce tumor growth factor \u03b2 that helps in EMT and metastasis .\u00a0InflammThe role of several inflammatory cytokines as prognostic markers is being studied. Higher levels of IL1 \u03b2, IL1Ra, IL18, and IL1\u03b1 in breast cancer tissues and significantly higher IL1 \u03b2 levels in stage II, III or IV breast cancers were reported . Higher The inflammatory cells acquire pro Vs antitumor capabilities depending on the interaction with TME and the macroenvironment. In addition to tumor-promoting inflammation, there are several studies in the last decade highlighting the bidirectional interaction between a cancer cell and microenvironment as well as macroenvironment that is key for a cancer cell to develop, survive, progress, and invade. Tumor exosomes play a major role in communication with TME and the macroenvironment to promote tumor growth and metastasis. Tumor exosomes modify stroma and immune cells creating a metastatic niche supporting the seeding of tumor cells .Tumor macroenvironment also referred to as tumor organismal environment includes metabolic, endocrine, lymphatic, hematopoietic, immunologic, microbiotic and neurogenic environments . MetabolCancer Epigenetics, Genome Instability and MutationEpigenetics is defined as heritable modifications in gene expression induced via changes in chromatin structure barring adjustments of DNA sequence ,45. AccuEpigenetic mechanisms that regulate chromatin structure can be divided into four essential categories - DNA methylation, covalent histone modifications, non-covalent mechanisms such as the incorporation of histone variants and nucleosome remodeling and non-coding RNAs together with microRNAs (miRNAs) ,50.The altered epigenetics of most cancers cells suggests that epigenetic therapies should have a fundamental clinical impact . The maihttps://www.nature.com/collections/afdejfafdb/Feb2020). One of the major studies of this project shows that each tumor had four or five driver mutation on average and at least one driver mutation was found in about 95% of the tumor samples compared with just 67% with exome sequencing [In 2011, Hanahan and Weinberg added genome instability into their list of fundamental characteristics of cancer, particularly as an enabling characteristic [quencing . Cancer quencing . Early oquencing . Howeverquencing . Endogenquencing . Recent quencing .Solid tumors of epithelial origin with extreme levels of genomic instability are associated with a potentially better prognosis compared with intermediate level . Also, cRecent fast progress in CRISP/Cas9 base editing technology has made it technically highly feasible to generate site specific nucleotide substitutions of DNA by manipulating highly intricate DNA repair pathway .An update to 2011 hallmarks of cancerSustaining Proliferative SignalingOne primary hallmark described by Hanahan et al is the cell's ability to become auto-sufficient and enable signals to sustain a continuous proliferative pathway . Some meThe mitogenic stimulus can be generated by an upstream receptor pathway or by downstream and intracellular circuits.Upstream circuits:\u00a0Tumor cells may induce the surrounding cells to support their growth with various growth factors ,63.\u00a0The Downstream cytoplasmic pathways may be GF independent and result in continuous activation of proliferation.a.\u00a0Somatic mutations trigger more downstream circuits:\u00a0As shown by Davies and Samuels, 40% of melanomas have some mutations in B-Raf protein result in continuous signaling generated by the activation of Raf to MAP- kinase (mitogen-activated protein).\u00a0Mutations in phosphoinositide 3-Kinase (PI3-kinase) isoforms may hyper-stimulate the signaling pathway ,65.b.\u00a0Disturbances of Negative-Feedback mechanisms that attenuate proliferative signaling:\u00a0Antiproliferative signals control and maintain cell homeostasis by inhibition of proliferation -69. FailThese mechanisms of turning off the negative control of cell proliferation probably contribute to drug resistance. The excessive proliferative stimulus may result in cell senescence or apoptosis -74. ThisEvading Growth SuppressorsCancer cell needs to avoid antigrowth signals to thrive . Growth Several other mechanisms, directly or not linked to pRb and p53 pathways, positively or negatively related to antiproliferative control, emerged in the last years and confirm the evasion of growth suppressors as an actual hallmark of cancer, helping to understand widely the complexity of this system, which can be a potential therapeutic target in many types of cancers.Alternative Reading Frame (ARF)ARF is a tumor suppressor protein encoded in the INK4b/ARF/INK4a gene locus located on chromosome 9p21 in humans that activate p53 in response to oncogenic signals, such as c- MYC . AlthougThe low expression of ARF mRNA is frequently observed in human cancers and is usually caused by hyper-methylation on the CpG island of the ARF promoter or deletion of the genetic region and has been described in breast, bladder, colon, liver, gastric, lung, oral, prostate and brain cancers and has emerged as a predictor of poor prognosis in breast, head and neck, colon and bladder carcinomas .GalectinsGlycosylation changes had emerged as an important process in cancer progression and among the glucan-binding proteins that deciphers the information encrypted by the glycoma, galectins had great importance . Gal7 shMelatoninMelatonin was found to induce phosphorylation of p53 at Ser-15 causing proliferation inhibition and prevention of DNA damage accumulation . The treMicro-RNAs (miRNAs)Many critical cell proliferation pathways involve miRNAs, dysregulation of which is responsible for evading growth suppressors and sustaining proliferative signaling in cancer cells. Some of these miRNAs interfere on E2F proteins expression, that are cell cycle regulators of cell proliferation, as miR-17-92 that inhibits E2F1 translation through breaking the positive feedback between c-Myc and E2F1  and was Other microRNAs negatively regulate CDK inhibitors, being needed to the cell cycle progression and cell proliferation, as miR-221/222, which has been identified to directly target p27Kip1 in glioblastoma cells  and thatLong Noncoding RNAs (lncRNAs)There are more than 10,000 lncRNAs in the human genome and they are implicated in almost all hallmarks of cancer, including acting as regulators of tumor suppressor genes and molecules that regulate the cell cycle . The lncResisting Cell DeathCancer formation involves different changes in the genome  and a seApoptosis pathways are classified into two types: extrinsic (receptor-mediated) and intrinsic (mitochondria-mediated). These pathways may be linked, molecules involved in one can affect the other one, and they can also be involved in other cell processes . The extThe regulation of apoptosis is also complex. The Bcl-2 family includes different proteins that influence apoptosis: some of them interact with mitochondrial proteins and prevent them from forming pores, inhibiting the release of apoptogenic factors, others neutralize the anti-apoptotic proteins ,112. IntThe main issue with conventional therapies in cancer treatment  is the evolution of adaptive mechanisms in cancer cells. Adaptive mechanisms may include upregulation of pro-survival proteins, suppression of pro-apoptotic proteins and defects in p53 signaling pathways. Many anticancer agents that target pathways involving deregulation of proteins like p53 lead to resistance, so direct targeting of mitochondria may be a promising strategy in attempts to restore the cells\u2019 ability to die -117.In conclusion, new drug targets can be identified and target selective therapeutic methods could be developed through analysis of apoptotic signaling pathways and apoptosis resistance mechanisms.Enabling Replicative ImmortalityThe ability of tumoral cells to achieve replicative immortality, allowing subsequently macroscopic growth, has been widely accepted as a hallmark of cancer . SeveralIn carcinogenesis, cells can activate mechanisms of telomere maintenance to overcome the cell senescence or apoptosis caused by telomere shortening. Several mechanisms of telomere maintenance have been identified and include telomerase gene hTERT promoter mutations , telomerAccording to data, in 85%-90% of cancer cells, the mechanism of telomere stabilization is reached by telomerase activation and only 5%-15% exhibit an ALT\u00a0pathway -126.Despite the upregulation of telomere reverse transcriptase (TERT) expression via promoter mutation -132 and Huang et al. showed that upon mitogen stimulation, not all but only a small subpopulation of T- cells reactivate telomerase and preferentially elongate short telomeres . It is pNevertheless, should the precise moment of telomeres shortening triggering telomerase activation be known, this could then have major effectiveness in stopping the tumor. This remains a challenge for future researchers .Inducing AngiogenesisAngiogenesis is a physiological process, that determines the formation of new vessels from preexisting ones. Angiogenesis is involved not only in embryonic development but also in damage and recovery. This process is tightly regulated and controlled by different mechanisms. By contrast, in pathological conditions (like cancer) angiogenesis is dysregulated and hyperactivated. The relationship between angiogenesis and cancer was first described in 1968 when several proangiogenic factors were discovered ,149.Tumor angiogenesis is a multistep process, and its main generator is hypoxia in tumor cells due to inadequate blood supply. It causes the production of angiogenetic factors such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), angiogenin, transforming growth factor \u03b1, TGF-\u03b2, tumor necrosis factor (TNF)-\u03b1 etc. by cancer cells, which bind to endothelial cell receptors of the vessels and initiate above mentioned process. Whenever the endothelial cells are stimulated, the secretion of matrix metalloproteinases (MMPs) is prompted, which causes degradation of the basal membrane. This process allows endothelial cells to invade surrounding tissues and start forming new vessels. In addition, factors such as angiotensin-1, -2, and their receptor Tie-2c are needed for the stabilization of newly formed vessels ,151.Angiogenesis plays a key role in cancer and according to many studies level of angiogenetic factors is correlated with tumor aggressiveness and has a strong predictive role ,152.Activating Invasion and MetastasisMetastasis is a hallmark of cancer and the cause of most cancer-related deaths . It is aThere are a variety of determining factors that govern the flexibility of a primary tumor to metastasize to different organs. These include genetic disorders to growth factors within the environment of the first tumor, the flexibility of tumor cells to detach from neighboring cells, the flexibility of tumor cells to digest the ECM and invade the vasculature. The tumor cells reorganize their cytoskeleton, express adhesion molecules on their surface to acknowledge adhesion sites, and chemotaxis or become migratory and motile resulting in loss of contact inhibition, and ultimately migrate to inappropriate locations giving rise to metastatic dissemination.In vivo as well as in vitro studies showed that metastatic tumor cells migrate individually . HoweverIn recent years, there has been a vital debate on whether EMT features a central role in cancer metastasis and resistance to chemotherapy . Some stAnother important thing that researchers are looking at is ECM. ECM is a dynamic and complex system that is composed of a wide spectrum of matrikines and cells that take part in invasion and metastasis .Our deep understanding of the dynamics of this hallmark will help us identify targets for molecular therapies which will halt or possibly reverse cancer growth and metastasis.Reprogramming of Energy MetabolismAbnormally high metabolic rates by cancer cells alter anti- tumor immunity by changing TME in the metabolic mechanism of glycolysis or amino acid metabolism. There are studies discussing the crosstalk between energy reprogramming in cancer cells and its association with antitumor immunity, and therefore suggest intervention of cancer metabolic agents provide an add-on benefit to cancer immunotherapy .Glycolysis and Lactate ProductionThe Warburg effect shows that tumors and cancer cells have increased rates of glucose uptake and lactate production, even in the presence of sufficient oxygen and low rate of oxidative phosphorylation . MetabolAmino Acids MetabolismTryptophan (Trp) and arginine (Arg) amino acids are considered to provide key nutrients in TME. Trp attenuates antitumor immunity in primary tumors and the neighboring tumor lymph nodes and Arg catabolism has been linked to suppression of antitumor immunity.Indoleamine 2,3-dioxygenase 1 (IDO1) enzyme is a rate-limiting enzyme in the metabolism of Trp in the peripheral tissues and IDOI inhibitors inhibit the first step of Trp catabolism . IDO1 isClinicalTrials.gov Identifier: NCT02903914). Epacadostat, an IDO1 inhibitor, is being studied in combination with pembrolizumab in patients with metastatic and/or locally advanced sarcoma (NCT03414229). Anti-IDO-1 agent (LY3381916) is also tested in combination with anti- PD-L1 checkpoint antibody (LY3300054) in solid tumors (NCT03343613). Additionally, a phase 2 study is ongoing to evaluate the activity of PD-1 inhibitor, nivolumab alone with and without IDO-inhibitor, BMS-986205, in patients with recurrent or persistent endometrial carcinoma or endometrial carcinosarcoma (NCT04106414). Given great and extensive interests of IDO inhibitors and other metabolic agents, it is expected there will be more clinical studies underway in addressing metabolic intervention in TME to aid on immunotherapy.PD-1 ligation and activation impairs metabolic reprogramming, including glycolysis and amino acid metabolism in T cells by inducing the expression of carnitine palmityl transferase 1A (CPT1A), a rate limiting enzyme of the fatty acid oxidation (FAO) pathway, and conversely, CTLA-4 inhibits glycolysis without augmenting FAO . ArgininEvading Immune SuppressionEvading antitumor immunity plays a major role in tumor progression and survival and must be considered as one of the hallmarks of cancer. Tumor escapes immune destruction by several mechanisms.\u00a0TGF\u03b2 plays a significant role in inhibiting T helper cell differentiation and promoting antitumor immunity ,172.\u00a0TumIn this era of immunotherapy, several studies are underway to improve anticancer therapy based on these mechanisms of evading immune suppression. Commensal microbiota was reported to have a role in improving anti-tumor response to immunotherapy and chemotherapy in extraintestinal tumors . BlockinIn conclusion, we summarized the hallmarks of cancer. Pro-survival autophagy is described as a new hallmark of cancer given its important role in tumorigenesis and potentially\u00a0an anti-cancer therapeutic target. The bidirectional interaction between a cancer cell and microenvironment as well as macroenvironment is key for a cancer cell to develop, survive, progress, and invade. In addition, cancer epigenetics, genome instability and mutation play a major role in enabling cells to acquire the hallmarks of cancer. Despite the advanced understanding, we have so far on hallmarks of cancer, there is much more to uncover in this field of research. Future research in this field is necessary to develop better ways to treat cancer."}
+{"text": "Adolescents should have access to high quality and responsive sexual and reproductive health, however, it is unclear to what extent the national policy on health and development of adolescent is implemented by health care workers in Plateau State. This study assessed the general availability of sexual and reproductive health services, the delivery of responsive adolescent sexual and reproductive health services and health care worker?s understanding of what constitutes adolescent responsive sexual and reproductive health services.Using a cross sectional design, we interviewed 409 health care workers selected through a multistage sampling technique, across six Local Government Areas of Plateau State, Nigeria using an interviewer-administered survey questionnaire.The most available sexual and reproductive health services was antenatal and delivery care (69.2%), contraception 25.9% and 14.9% reported post abortion care. Only 1.2% indicated the availability of the four recommended essential sexual and reproductive health services  /HIV and post abortion care) in their facilities. Little over half (58.4%) felt their facilities were adequate in meeting the sexual and reproductive health needs of adolescent and this was associated with delivery of post abortion care  and providing sexual and reproductive health services to adolescents without parental consent . Most health care workers had poor understanding of adolescent responsiveness of sexual and reproductive health services, understanding better among health workers who provided services without parental consent and in a separate room for privacy and confidentiality.We conclude that adolescent sexual and reproductive health services is not yet as stipulated in the national policy in Plateau State, Nigeria and in general, health workers have poor understanding of what it means to provide adolescent-responsive services.The online version contains supplementary material available at 10.1186/s12905-023-02288-1. Access to comprehensive sexual and reproductive health (SRH) information and other services is a basic right of adolescents, as acknowledged at the 4th International Conference on Population and Development (ICPD) in Cairo in 1994, and enshrined in the Rights of the Child . These iAccording to the policy, adolescents should be provided scientifically accurate SRH information on sexuality, menstrual hygiene, prevention of unintended pregnancy and prevention of STI and HIV , 9. Furtadolescent responsive [All SRHS should be delivered in a non-judgmental manner and in an environment that respects the rights and privacy of adolescents  , 11\u201313. sponsive \u201316. One sponsive \u201320.To ensure access to SRHS and improve adolescent SRH health seeking, adolescent health services were integrated into the primary healthcare (PHC) centres, which are found in all communities and are often closer to where adolescents spend most of their time , 15. We While geographical and financial constraints may impede adolescent\u2019s utilization of SRHS, prior research has also shown that the negative attitudes of HCW and their non-responsiveness to the needs of adolescents, impedes adolescents\u2019 SRH care seeking \u201327. AlsoA cross sectional survey was conducted among healthcare workers in PHC facilities in six selected Local Government Areas (LGAs) in Plateau State, north-central Nigeria. Every LGA has an average of 34 PHCs that provide basic health services, which include health promotion/education and treatment of common illnesses, in addition to providing SRHS. A diversity of HCWs provided consultation services at PHC, including nurses, community health extension workers, environmental health officers, laboratory technicians and volunteers. Although consulting health services at PHC should be delivered by doctors, nurses, midwives or medical assistants, due to a shortage of these types of staff members in PHC in Plateau State, workers from other cadres are also tasked with providing consulting health services.The participants were recruited through a multistage sampling technique. We began by selecting six LGAs from the 17 LGAs in Plateau State. All the 230 PHC facilities across these six selected LGAs were included in the study. Of an estimated 690 HCWs in the selected PHCs, those who never provided any consulting services were excluded, resulting in a total of 446 HCWs who were eligible for participation in the study. Out of these, 409 (91.7%) HCWs completed the survey. All HCWs provided informed consent before commencing with the survey.Eight trained volunteer resident doctors from Jos University Teaching Hospital (JUTH) administered a self-report questionnaire through face-to-face interviews. Questions about SRHS available and provided to adolescents reflected the national policy on adolescent and young people . QuestioDemographic and professional characteristics: The demographic and professional information collected included: age, sex, marital status and education, specialty of healthcare workers, years of working and if trained on adolescent SRH.Availability of SRHS: The HCWs were asked about the SRHS generally available in their health facilities: contraceptives (yes/no), pregnancy testing (yes/no), STI/HIV testing (yes/no), STI/HIV treatment (yes/no), post abortion care (yes/no) and antenatal and delivery care (yes/no).Provision of ASRHS: To assess if SRHS were provided to adolescents as stipulated in the national policy, HCW were asked if they provided the following SRHS to adolescents: SRH counselling (Yes/No), contraceptives (Yes/ No), STI treatment (Yes/No) and post abortion care (Yes/No). They were also asked if, as also stipulated in the national policy, ASRHS were provided in separate/private rooms to ensure privacy and confidentiality (Yes/No) and were provided without parental consent (Yes/No).HCWs were asked if they thought their facilities had adequate space, equipment and commodities to provide SRH meeting the needs of adolescents (Yes/No).Understanding of adolescent responsiveness of SRHS: HCW were asked; \u2018SRHS are adolescent-responsive when\u2026.\u2019 and choose one or more of the following response options: 1) all SRHS are made available and accessible to adolescents, 2) the services are provided free or at affordable cost, 3) services are provided without being judgemental and 4) services are provided in separate rooms for adolescents to ensure privacy and confidentiality\u2019. Each of these options represents an indicator of adolescent responsiveness. Every option chosen was given a score of 1 and a summary score was calculated to reflect understanding of adolescent responsiveness (range: 0\u20134).The data analysis was conducted using IBM SPSS version 23 . Data was cleaned and all incomplete entries were removed before the analysis.We calculated descriptive statistics for HCWs responses regarding the availability of SRHS in their facilities, SRHS provided to adolescents and delivery of ASRHS, perceived adequacy of facilities in meeting the adolescents\u2019 needs and understanding of adolescent responsive SRHS.p \u2264 0.05.Subsequently, we ran univariable (not shown) and multivariable logistic regression analyses to assess associations between the demographic and professional characteristics of HCW and the provision and delivery of ASRHS. We also conducted univariable (not shown) and multivariable logistic regression analyses to assess associations between the perceived adequacy of the facilities of HCW in meeting the needs of adolescents and the demographic and professional characteristics of HCW as well as the specific ASRHS provided. We conducted univariable (not shown) and multivariable linear regression analyses to assess associations between the demographic and professional characteristics of HCW and their understanding of adolescent responsiveness. For all analyses of covariates, the level of significance was set at Participants were mostly women (66.3%), above the age of 40 years 53.1%) and married (89.5%); nearly all (94.9%) did not have a university degree (94.9%). Also, the majority were nurses or CHEW (79.5%). The others (20.5%) were environmental health officers, laboratory technicians or volunteers. Just over (53.6%) had 1\u201315 years working experience, nearly all (90%) had never had any training on adolescent SRH  reported that some SRHS were available in their facilities. The most frequently mentioned SRHS available was antenatal care and delivery services (69.2%), followed by STI treatment (53.6%). Only 6.9% of the HCW indicated that STI testing was available in their facilities and treatment of STI was mostly based on symptoms and not on testing. Only 25.4% of HCW indicated they had contraceptives available in their health facilities, while merely 14.9% said post abortion care was available in their health facilities.ASRHS most frequently provided was counselling, predominantly on abstinence (67%). Only 31.5% of the HCWs indicated that they provided counselling to adolescents on SRH issues other than abstinence, STI treatment was provided to adolescents by 53.6% and 15.2% provided post abortion care.According to the HCW, 17.6% of the facilities did not provide any of the four SRHS for adolescents stipulated in the national policy , 44% provided one service, 27.2% provided two services, 10% provided three services and only 1.2% provided all four services. Also, most participants (71.2%) said their facility did not provide SRHS without parental consent and 32.8% provided ASRHS in separate rooms. Their facilities were perceived as adequate in meeting the SRH needs of adolescents by 58.4% of the HCWs. Regarding understanding of adolescent responsive SRHS, 17.8% of participants did not mention any, 79.7% mentioned only one, 1.7% mentioned two, and 0.7% mentioned three. None correctly identified all four aspects of adolescent responsiveness.Multivariable logistic regression analyses showed no significant association between the demographic and professional characteristics and SRHS provision, except for HCW gender, males were more likely to provide STI treatment   and providing SRHS to adolescents without parental consent  , male \u03b2\u2009=\u20090.074), unmarried (\u03b2=-0.008), nurses/CHEW (\u03b2\u2009=\u20090.032), held a university degree (\u03b2\u2009=\u20090.010), had more years of work experience (\u03b2\u2009=\u20090.068) and had received training on ASRH (\u03b2=-0.008). Understanding of adolescent responsiveness was also higher among HCWs who reported providing counselling (\u03b2\u2009=\u20090.067), contraceptives (\u03b2\u2009=\u20090.025) and not requiring parental consent (\u03b2\u2009=\u20090.275). In contrast, understanding of adolescent-responsiveness was lower amongst HCW who reported providing STI treatment (\u03b2=-0.033). (See Table\u00a0, unmarriNigeria has recently made efforts to ensure that adolescents have access to high quality and responsive SRHS, by developing a national policy and integrating SRHS into primary healthcare facilities. However, one frequently cited hindrance to utilization of SRHS by adolescents is the non-responsive attitude of HCWs \u201327. We fOne fundamental requirement to providing adolescent responsive SRHS is the capacity of the providers. It is expected and stipulated in the Nigerian policy, that providers should be qualified and trained to provide evidence-based services, especially due to the sensitive nature of the matter for young people , 33, 35.Most HCWs reported SRHS to be available in their facilities and the most frequently mentioned services were antenatal and delivery care, followed by STI treatment\u2013 which was generally based on symptoms with no testing. Nonetheless, these services did not cater for the range of adolescent SRH needs and were not adolescent focused , 39. TheSRHS delivery is considered to be adolescent responsive when the recommended SRHS are provided without being judgmental and in an environment that respects the privacy and confidentiality of adolescents. We found that almost all HCWs who provided SRHS to adolescents, including those who provided STI treatment and post-abortion care, often did not ensure adolescents\u2019 privacy by providing services in a separate room where they could not be seen and heard by other health service attendees. Also, HCW generally provided ASRHS only with parental consent. Privacy and confidentiality are key aspects of adolescent responsive SRHS delivery because adolescents do not appreciate discussing their sexual and reproductive health concerns in the presence of adults, including their parents , 44. TheWe observed that the delivery of ASRHS was not significantly associated with any of the demographic and professional characteristics of HCWs, except for gender, with men being more likely to provide STI treatment. Having ever received training on ASRH was not significantly associated with providing any of the SRHS. However, this finding may be due to the small number of HCWs in our sample that had received such training. Therefore, these results should not be taken to suggest that training does not contribute to the ability of HCW to provide adolescent responsive SRHS, especially since other studies have highlighted that training resulted in more favourable attitudes towards providing ASRHS , 48, 49.In view of the limited provision of ASRHS reported by HCW and the non-adolescent responsiveness of ASRHS provision, it is noteworthy that some HCW felt their facilities adequately met the SRH needs of adolescents. Reflecting the national policy, HCWs who perceived adequacy in meeting the needs of adolescents were also more likely to offer recommended services, notably, post-abortion care and to provide SRHS without parental consent. It should be noted that provision of these ASRHS is contested, reflecting religious and personal beliefs that may affect ASRHS. Provision of SRHS is already generally rare, and HCW normally do not provide such services to adolescents, believing they are minors and must have the approval of their parents or guardian before seeking care , 31, 47.To the best of our knowledge, this study it is the first to assess the extent of implementation of the Nigeria national policy on the responsive delivery of SRHS for adolescent and young people. Furthermore, the focus on HCWs perspectives of adolescent responsive SRHS delivery is innovative and provides novel insights into some of the possible reasons for the poor delivery of ASRH. The findings go beyond what is already known and offer guidance for policy and programmatic redirection. Additionally, we made use of a robust sampling frame and stepwise approach to recruit a sample of HCWs to optimally reflect the diversity of these professionals in Plateau State, Nigeria. We also acknowledge the limitations of our study; including the cross sectional design, which precludes any causal inferences. Furthermore, the study was conducted only in one state in Nigeria, limiting the generalizability of the findings to the entire country or other countries in the region. Also, data were collected through an interviewer-administered self-reported questionnaire, and responses may have been affected by recall bias as well as social desirability bias.Delivery of adolescent responsive SRHS in Plateau State, Nigeria is not as set out in the national policy. This study shows that most HCW at PHC do not deliver the full range of ASRHS and do not deliver ASRHS in an adolescent responsive manner. Encouragingly, the perception of HCW regarding their services being adolescent responsive was higher when they actually delivered relevant ASRHS. In general, the HCWs in this study, however, had poor understanding of adolescent responsive SRHS. To ensure delivery of adolescent responsive SRHS, we recommend scaling up appropriate training for HCW in Plateau State to improve their knowledge and skills in providing quality SRHS to adolescents who need it. However, although knowledge and training are essential, our results also indicate that this is likely not sufficient to provide responsive ASRHS in the absence of structural facilitating factors, such as private consulting rooms, which help ensure that adolescents experience a sense of safety and privacy to freely discuss SRH issues.Below is the link to the electronic supplementary material.Supplementary Material 1"}
+{"text": "Ebola virus disease is a medical condition whose consequent effects on quality of life of patients. In the history of infectious diseases, there have been pathologies that have had significant repercussions for caregivers, healthcare providers and the community.This study investigate determinants of quality of life among caregivers of adolescent and young adult Ebola survivors in Democratic Republic of the Congo.p-value. Statistical significance was defined as a p-value 0.05. The final multivariate model contained variables that were significant in the bivariate analysis. Prior to data collection, a research permit from National Ethical Committee of Research in Democratic Republic of the Congo\u00a0was obtained. Written informed consents from literate or illiterate caregivers of adolescent and young adult Ebola survivors were obtained. Throughout the study, participants' privacy and confidentiality were respected.This was a cross sectional study. The study sites were the two health districts of Beni and Katwa, in North-Kivu province in the Eastern part of Democratic Republic of the Congo. The study period was from April to August 2022. Participants of the study were caregivers of adolescents and young adult Ebola virus survivors. Simple random sampling technique was used to select the 68 study participants. A questionnaire was administered. Data was collected using pretested questionnaire of WHO quality of life Bref (WHOQOL-BREF) and CommCare by Dimagi.Inc. lastest Version 2.52.1\u00a0and\u00a0 a sum of score\u00a0of 78 or higher indicated a high level of life quality. To determine the quality of life of caregivers of adolescents and young adult EVD survivors, descriptive analysis was used. The Pearson correlation coefficient was utilized to check whether the predictor variables are multicollinear. The regression analysis produced the crude odds ratio (COR), adjusted odds ratio (aOR), 95% confidence interval (CI), and p\u2009=\u20090.02); OR:(95% CI), 3.17: (1.2 \u2013 8.36), With regards to place of residence, caregivers who lived in town were less likely to have good quality of life compared to those in rural (p\u2009=\u20090.01); OR: (95%CI), 0.25: (0.09 \u2013 0.72).A total of 68 care givers participated in the study, with a majority 54/68(79.41%) having poor quality of life. Men were 3.17 times more likely to record good quality of life than women (The quality of life of caregivers of adolescent and young adult Ebola survivors in Democratic Republic of the Congo is poor. To be woman caregiver and to live in town are\u00a0determinants associated with poor quality of life among caregivers of adolescent and young adult Ebola survivors. In Democratic Republic of the Congo, Ebola Virus disease was first encountered in 1976. Since then, there were more than 14 outbreaks in the region, with the frequency of occurrence increasing drastically over time . DespiteFor this reason, the need for caregivers to manage the disease and support these survivors have intensified , 4. CareStudies have been conducted on the quality of life of caregivers of patients with chronic illnesses , 8. AddiThis was a cross-sectional study.The study was conducted in the health districts of Beni and Katwa in North Kivu, Democratic Republic of the\u00a0Congo (DRC). North Kivu is one of the provinces in Eastern part of DRC and has a population of about 1.9 million. It borders Lake Kivu.The study was carried out during a 4\u00a0months\u2019 period from April to August 2022.Participants of the study were caregivers of adolescents and young adult Ebola virus survivors. Random sampling technique was used to select the 68 study participants.Caregivers of adolescents and young adult Ebola virus survivors were enrolled when they stayed for 4\u00a0months with adolescents or young adult Ebola virus survivors in the study setting.Caregivers had a thorough history and a complete physical examination undertaken to capture their social demographics, co morbidities as well as the bio-medical characteristics, using the designed questionnaire for data collection. Further information regarding other chronic illnesses  as well as chronic medication was also obtained through reviewing the caregiver\u2019s medical records. Severely ill caregivers with chronic health problems and who were unable to provide adequate information were excluded to the study.The provision of written informed consent was required for all study participants before enrolment into the study.2 x p (1-p)/e2.A total sample size of 68 participants was estimated using the Kish Leslie (1965) formula for finite populations, based on determinants of quality of life among adolescent and young adult Ebola survivors in Democratic Republic . The forData was collected using CommCare by Dimagi.Inc. Version 2.52.1. Sociodemographic, community and medical variables were collected. Socio-demographic variables included; age (25\u201355 and\u2009>\u200955), sex, education level , marital status , residence , religion  and socio-economic status. The medical variables were muscle pain, chest pain, fatigue, sleep problems. Community variables wee social support, stigma, experienced stress. Data was collected using the interviewer administered pretested questionnaires of WHO quality of life BREF (WHOQOL-BREF). A total score\u2009<\u200978 was regarded as poo quality of life and a sum of\u2009\u2265\u200978 indicates good quality of life , 11. A LData were collected by trained research assistants who interviewed the caregivers of adolescents and young adult EVD survivors.p-value. The final multivariate model contained variables that were significant in the bivariate analysis. The research used the assumption that each pair of outcomes had a proportional chance of being either bad or good quality of life. The factors with P-values less than 0.05 were considered significant.Data was entered using CommCare by Dimagi.Inc. lastest Version 2.52.1 and exported into STATA version 12 for analysis. Descriptive analysis was used. Categorical variables were analysed using frequencies, proportions and percentages. The regression analysis produced odds ratio (OR), 95% confidence interval (CI), and Prior to data collection, a research permit from National Ethical Committee of Research in Democratic Republic of Congo and Great Lake University of Kisumu-Kenya, helped to alleviate mistrust and allowed the participants to reveal much of the information required for the study. Written informed consents were obtained from literate or illiterate caregivers of adolescent and young adult Ebola survivors before enrolment into the study. Throughout the study, participants' privacy and confidentiality were respected.Out of 68 caregivers who participated in the study, 48.5% (33/68) were females and 51.5% (35/68) were males. The mean age(\u00b1\u2009SD) of the participants was 46.68(16.17) years. A majority were between the age 25\u201355\u00a0years 63.2% (43/68) and\u2009>\u200955\u00a0years 36.8% (25/68). Catholic religion was the most dominant religion 54.41% (37/68) followed by protestants 30.88% (21/68), see Table Seventy-four percent (54/68) of study participants had poor quality of life while 20.59% 14/68) had good quality of life, see Fig.\u00a04/68 had A total of 68 caregivers participated in the survey, with a majority 79.41% (54/68) having poor quality of life while 20.59 % (14/68) having good quality of life, see Table In terms of medical factors, results from the table show that, approximately, 25. % (17/68) have fatigue; 23.53% (16/68) have muscle pain;17.65% (12/68) have sleep problem and 13.24% (9/68) have chest pain. With regards to community factors, almost three quarter of the caregivers who participated in the study 85.29% (58/68) had experienced stress, 70.59% (48/68) had experienced stigma and 58.82% (40/68) reported no social support, see Table p = 0.01), see Table\u00a0Markers of sex and occupation were determinants associated with quality of life on bivariate analysis (p = 0.619), see Table\u00a0Muscle pain and experience of stress were not determinants associated with quality of life on bivariate analysis ((p\u2009=\u20090.02); OR:(95%CI), 3.17: (1.2 \u2013 8.36), caregivers who lived in town were less likely to have good quality of life compared to those in rural (p\u2009=\u20090.01); OR:0.25: (0.09 \u2013 0.72) see Table On multiple analysis, sex and place of residence significantly influenced quality of life of caregivers of adolescents and young adults Ebola survivors, male caregivers were 3.17 times more likely to record good quality of life than female caregivers 1.2 \u2013 8.3, caregivThe purpose of this study was to investigate determinants of quality of life among caregivers of adolescent and young adult Ebola survivors in the Democratic Republic of the Congo. This study reported a high prevalence of poor quality of life among caregivers, with three quarters having poor quality of life. This was extremely high compared to findings from a study in Uganda on QOL of caregivers of cancer patients, which reported less than half of the caregivers to have poor quality of life. This kind of divergence could be explained by the difference in the dynamics of the diseases, geographical regions and economic abilities of the two nations, given the long history of political instability in the DRC. Additionally, the effect of Ebola virus on its survivors and caregivers is profound. Given its severity, caregivers suffer psychological distress as a result of fear of infection, or loss of a loved one . FurtherSex of the caregivers was a significant determinant of their QOL in this study. Men were highly likely to have good quality of life than women. The gender discrepancy in viewpoints of illness perception sequence may contribute to female caregivers\u2019 lower level of quality of life than men.This finding is consistent with findings from studies by Sun Young et.al. and Shao-Yin et al., which also reported men to have higher QOL than women caregivers , 15. AnoIt was also established from the study that place of residence had significant impact on QOL of caregivers of Ebola survivors. Inherently, people\u2019s interaction with their residential environments plays a vital role in their health. Their interpersonal and social associations in these environments influences their quality of life . Urban rThe study found no significant association between QOL and marital status, occupation, social support and monthly allowance of caregivers. However, there is a substantial number of studies that have reported association between education, monthly income, social support and QOL of caregivers , 20, 21.The study found no significant association between QOL and marital status, occupation, social support and monthly allowance of caregivers. However, there is a substantial number of studies that have reported association between education, monthly income, social support and QOL of caregivers , 20, 21.Three quarter of caregivers of adolescents and young adult Ebola virus survivors have poor quality of life and being women caregiver and living in town are associated determinants with poor quality of life among caregivers of adolescent and young adult Ebola survivors in Democratic republic of the Congo."}
+{"text": "Candida is the largest genus of medically significant fungi. Although most of its members are commensals, residing harmlessly in human bodies, some are opportunistic and dangerously invasive. These have the ability to cause severe nosocomial candidiasis and candidemia that affect the viscera and bloodstream. A prompt diagnosis will lead to a successful treatment modality. The smart solution of biosensing technologies for rapid and precise detection of Candida species has made remarkable progress. The development of point-of-care (POC) biosensor devices involves sensor precision down to pico-/femtogram level, cost-effectiveness, portability, rapidity, and user-friendliness. However, futuristic diagnostics will depend on exploiting technologies such as multiplexing for high-throughput screening, CRISPR, artificial intelligence (AI), neural networks, the Internet of Things (IoT), and cloud computing of medical databases. This review gives an insight into different biosensor technologies designed for the detection of medically significant Candida species, especially Candida albicans and C. auris, and their applications in the medical setting. Candida species alongside SARS-CoV-2 have brought the problem to the foreground. Even the World Health Organization (WHO) was compelled in late 2022 to publish the fungal priority pathogens list (FPPL) for the first time\u2014a catalog of 19 fungi that threaten public health [Fungal pathogens had long been relegated to the \u201cdungeons\u201d of the modern healthcare system as compared to bacterial pathogens. However, the outbreaks of infections by Candida spectrum include Candida albicans, Candida auris, Candida krusei, Candida tropicalis, Candida parapsilosis, Candida rugosa, Candida guilliermondii, and Candida glabrata [C. albicans is a commensal microbe and part of normal human flora, residing harmlessly on the skin and inside the body, in locations such as the mouth, gut, throat, and vagina. However, it becomes an opportunistic invader in immunocompromised or immunodeficient individuals. An invasive disease caused by any Candida species is termed candidiasis. Candidiasis may occur in individuals due to waning immunity, pH change, diabetes, surgical procedures, antibiotic therapy, neutropenia, parenteral nutrition, malignancy, or HIV, and in neonates [The medically important species of the glabrata . C. albineonates ,5. Orophneonates .Candida infections are quite common in hospitalized patients. The mortality rate among candidemia patients ranges from ~20 to 40% [C. auris, a multidrug-resistant pathogen, causes serious bloodstream and wound infections in hospitalized patients, especially those having breathing tubes, feeding tubes, or central venous catheters [Bloodstream 0 to 40% ,8,9,10. atheters .Candida infections. However, resistance patterns are on the rise, with C. auris being among the most significant multidrug-resistant species, while C. krusei and C. glabrata are intrinsically resistant to echinocandins [Azoles, echinocandins, and amphotericin B drugs are commonly used to treat ocandins ,13,14,15The timeliness of therapy is a victim of insensitive and slow diagnostic tools like the fungal culture method, microscopy, serology, and histopathology . This de\u221212 M), femtomolar (10\u221215 M), and attomolar ranges (10\u221218 M). Hence, next-generation approaches based on molecular techniques such as polymerase chain reaction (PCR), DNA-sequencing-based methods, protein fingerprinting by matrix-assisted laser desorption/ionization\u2013time-of-flight mass spectrometry (MALDI-TOF-MS), and ultrasensitive laser-based technologies that target specific Candidal biomarkers [. will aid in this direction [Sudden fungal outbreaks can be tackled with revolutionary concepts in the field of biosensors with sensitivity down to picomolar 10\u2212 M, femtoirection . The funirection . A microanges 10\u2212 M. HenceCandida infections, such as blood culture and microscopy, have several limitations that can lead to delayed or inaccurate diagnosis. These methods are time-consuming, require skilled personnel, and may not be sensitive enough to detect low levels of Candida species cells in clinical samples [Candida species [Candida infections [Candida species. Biosensors are the appropriate tools in this approach. Generally speaking, biosensors offer rapid and accurate detection of Candida species, which can lead to earlier diagnosis and treatment; can be more cost-effective than traditional diagnostic methods; can be used for real-time monitoring of circulating analytes, which can reflect the efficacy of antifungal treatment; and can be designed to be portable and easy to use, making them suitable for point-of-care diagnosis [Traditional diagnostic methods for  samples . In addi species . Furtherfections . Therefoiagnosis ,23.Candida surveillance by different types of biosensors, focusing on the available literature published until now to the best of our knowledge, emphasizing the impact of modern technologies and their applications in the medical setting.This review aims to give an update and call for more attention in regard to medically important Candida antibodies are applied for detecting mannan antigen and anti-mannan antibodies, respectively, in infected serum [Candida biomarkers exploited for diagnostic tests are depicted in Diagnostic and commercial tests for invasive candidiasis have been developed, based on serological markers like mannan antigen, anti-mannan antibodies , the anted serum . PotentiMannan is a major cell wall component and is the primary circulating antigen encountered during infection. Enolase  and arabCandida Plus agar prominently distinguish Candida auris. Direct microscopy to locate Candida albicans in clinical samples like sputum, swabs, tissue biopsies bronchoalveolar lavage, cerebrospinal, and other body fluids is faster as compared to the culture method. KOH, Calcoflour White, Chicago Sky blue 6B, Blankophor, Gram stain, and India ink are used for direct microscopy [Traditional approaches for detection are mostly based on culture methods, microscopy, serology, and histology. In vitro culture methods on selective or indicator growth media such as CHROMagar\u2122 croscopy . Howevercroscopy .Candida can be identified by histology, which is a better and more cost-effective tool of choice for invasive fungal detection than the culture method. Periodic acid\u2013Schiff (PAS) and Gomori\u2019s methenamine silver (GMS) stains are specially used [Histoplasma and small Candida species (C. glabrata) [The yeast, pseudohyphal, and hyphal polymorphic features of lly used ,26. Somelly used . Also, mlabrata) . Histololabrata) ,26. The Candida antigens or circulating antibodies in the blood, like cell wall mannan glycoprotein antigen and anti-mannan antibodies. PLATELIA\u2122 Candida Ag Plus 62784 (Bio-Rad Laboratories) and SERION ELISA antigen, Hemkit Candida Ab test detect circulating mannan antigen qualitatively and quantitatively in human serum or plasma [Serological tests detect the presence of r plasma . Interfer plasma .No single technique is sufficient to provide accurate and specific information for species identification and antifungal resistance. It is better to rely on a combination of traditional and molecular techniques like PCR, sequence-based identification using peptide nucleic acid fluorescence in situ hybridization (PNA-FISH), and matrix-assisted laser desorption/ionization (MALDI) spectrometry simultaneously ,29.The clinical symptoms of candidiasis are non-specific and appear later in the course of infection. In addition, nearly one-third of candidiasis patients do not receive positive blood culture reports, and results are obtained at approximately 48\u201372 h or more. Species identification takes even longer. Its sensitivity in hematogenous disseminated candidiasis is <50% .C. auris outbreaks during the COVID-19 pandemic. Nosocomial infections, invasiveness, and high fatality rates seriously challenge the development of fast, reliable, and accurate detection systems for Candida species. Biosensor technology is a promising step in this direction to reduce medical costs and fatalities. Early diagnosis with biosensors will reduce medical burdens. Also, biosensors enable continuous monitoring opportunities to assess the response of the patient toward treatment modalities [Prompt surveillance systems are of utmost importance to counter the rapid, sudden rise and spread of fungal infections worldwide, viz., dalities .A biosensor is a bio-analytical device that translates information from biological or chemical reactions into a quantifiable signal proportional to the strength of the reaction. It has three components\u2014a biorecognition element, a transducer, and a signal processor. The quality of a biosensor depends upon its sensitivity, selectivity, reproducibility, reusability, detection limit, and response time. Sensitivity is the relationship between change in analyte concentration and signal intensity at the transducer. Selectivity is its ability to bind and respond only to the target analyte molecule and none other. Reproducibility is the ability to produce matching results in response to the target analyte each time the same/identical biosensor is used. Reusability is the ability to use the biosensor many times. The detection limit is the lowest analyte concentration that can generate a signal. Response time is the time required by the device to elicit a signal following the biochemical reaction.Biorecognition Element: This element translates signals from a biochemical reaction involving receptor molecules and analyte molecules into a chemical or physical output signal. This is involved in the biosensing event. The input signal is proportional to the analyte concentration. Enzymes, antibodies, nucleic acids, aptamers, hormones, microbes, molecularly imprinted polymers, phage display proteins , and nanTransducer: A biosensor is categorized by the type of transducer. Transducers convert signals from the biorecognition element to measurable signals. These are connectors between the biorecognition unit and the signal processor. The biomolecule is immobilized on the transducer surface by covalent attachment, adsorption, cross-linking, entrapment, and micro-encapsulation. Transducers with high-affinity binding generally give a one-time signal, whereas low-affinity binding allows continuous monitoring of analyte concentrations . TransduSignal Processor: The signal processor converts the measurable signal from the transducer into an interpretable and quantifiable signal. The output signal from the transducer in the form of current is converted into voltage and further processed for noise removal through various filters by the processor .The immobilization of analyte biomolecules on the sensor surface of the biorecognition element is one of the key steps of biosensor fabrication. The biomolecules may be antigens, antibodies, peptides, enzymes, nucleic acids, aptamers, or whole cells. The immobilization is essential to stabilize the biomolecules under diverse conditions of temperature, pH, hydrophobicity, oxidizing environment, and solvent polarity. This is important for the reusability of the biosensor. Immobilization brings the analyte molecules in close proximity to the transducer. The process should be such that the active site of the molecule remains accessible for interaction while maintaining bioactivity and conformational stability. Immobilization methods may be reversible or irreversible.Reversible Immobilization: In this method, the adhered biomolecules can be detached from the surface under mild conditions to regenerate the sensor matrix for the reusability of the system. The two ways under this process are as follows:Adsorption: It is a physical process involving hydrogen bonds, hydrophobic interaction, Van der Waal\u2019s forces, and salt linkages. Molecules bind to the surface with low affinity, and hence the process may be reversed by variations in pH, temperature, etc., to affect the sensitivity and reproducibility. Examples include whole-cell adsorption and immunoadsorption.Affinity Binding: Affinity binding involves non-covalent interactions between the activated support and a specific group on the biomolecule. Examples include antigen\u2013antibody, lectin\u2013sugar, streptavidin\u2013biotin, and Protein A/G with the Fc region of the antibody. Since this natural immobilization occurs at high concentrations but with high sensitivity, this process is useful for microarray applications .Irreversible Immobilization: The biosensor molecules irreversibly adhere to the sensor matrix.Cross-linking: Here, biomolecules are linked to the transducers via cross-linking molecules with free reactive ends that form covalent bonds with functional groups. Whole cells and enzymes can be immobilized on the electrodes by being dipped in cross-linkable polymers such as glutaraldehyde and glyoxal.Covalent binding: This binding is the most exploited, is stable but generally irreversible, and is stronger. It is achieved through functional groups like amino, hydroxyl, carboxylic, and thiol. It provides a homogeneous and dense distribution of the sensor molecules on the sensing surface to improve sensitivity. DNA immobilization with covalent bonding generally involves linker functional groups. Due to its specificity, biomolecular leakage is evaded here. Examples include 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC), a carboxyl activating agent, and N-hydroxysuccinimide (NHS) for coupling carboxyl groups to primary amine groups, yielding reactive NHS esters .Entrapment: Here, the biosensing molecule is enveloped within a polymeric network while allowing substrates and products to pass through. This is a simple way to encapsulate, absorb, or localize the analyte molecules into the sensor matrix. It is achieved either by electrochemical polymerization of the polymer on the transducer surface or layer-by-layer deposition . EntrapmCandida species , leading to a sequence of oxidation and reoxidation reactions involving a ferrocene derivative and ferricyanide ions generating an electric current [Candida antibody-functionalized sensor specifically, accurately, and rapidly detects the presence of Candida using membrane-based electrochemical impedance spectroscopy. The sensitivity of the biosensor was up to the clinically relevant level of 10 CFU/mL [ current . Similar0 CFU/mL .Candida species\u2014C. albicans, C. tropicalis, C. krusei, and C. glabrata\u2014based on their ploidy has been demonstrated by an electrochemical AMP-based biosensor. The sensor platform is composed of self-assembled nanofilms of electropolymerized poly(thiophene acetic acid) (PTAA) and amino-functionalized TiO2 nanoparticles (NH2-TiO2 NPs) and presented the highest electrochemical response when binding was analyzed using the electrochemical impedimetric technique [Sensitive and fast detection of echnique .C. albicans was designed in 1986 by the Japanese group of Muramatsu et al. [6\u20135 \u00d7 108 cells cm\u22123. Anti-Candida antibodies were immobilized on the surface of anodically oxidized palladium-plated electrodes. A quartz piezoelectric crystal sensor was dipped into the Candida suspension to measure the increment in surface mass due to immunoadsorption of the microbial cells and the decrement in the resonance [A piezoelectric biosensor records the oscillation change due to the binding event on a piezoelectric crystal. A piezoelectric immunosensor for u et al. . The detesonance .Optical biosensors are the most intuitive biosensors of all the categories. Here, biomolecular concentration in a test sample is measured by measuring the change in optical signals like reflection, refraction, diffraction, absorption, fluorescence, and chemiluminescence. In optical biosensors, optical transducers that detect the changing intensity of absorbed or emitted light proportional to that of quantum change in the biorecognition element are exploited. These have label-free molecules, high specificity, economic production costs, and a small footprint.C. albicans with the detection limit of 32 CFU/mL, by binding to mannan on its surface. Two-dimensional particle spacing in the crystal is reduced due to cross-linking, causing a blue shift of the diffracted light. This visual diffraction shift is either measured with a spectrometer or determined from the Debye diffraction ring diameter.Two-dimensional arrays of photonic crystals embedded in Con A hydrogels selectively identify Diagnosis of oral candidiasis in smears and swabs is rapidly performed by fungal fluorescent staining as well as with periodic acid\u2013Schiff stain and observation under a fluorescence microscope . The patCandida species\u2014C. albicans, C. parapsilosis, C. glabrata, and C. tropicalis\u2014involved in vulvovaginal candidiasis. The designed system involves a sample-processing cassette with a nucleic acid analysis device. Nucleic acids are released within 15 min by a vibrating injector and grinding glass beads and analyzed within 30 min. Specific primer sets were introduced for each species. The technique used is the loop-mediated isothermal amplification (LAMP) method. The sensitivity is <2 CFU/reaction with a very low sample size of 1.41 \u00b5L [A portable, rapid multi-target system for nucleic acid analysis devices has been fabricated for the simultaneous detection of four  1.41 \u00b5L .C. parapsilosis infections, a reliable DNA detection assay has been developed by combining LFS with recombinase polymerase amplification (RPA). The sensitive and specific detection utilizes the catalytic subunit 2 of the \u03b2-1,3-glucan synthase (FKS2) gene of C. parapsilosis, amplified by a primer\u2013probe set with base mismatches (one base modified by the reverse primer and four bases modified by the probe). The rapid amplification and the appearance of a pink test line of the FITC and biotin-labeled FKS2 gene on the strip takes only 30 min at 37 \u00b0C. The efficiency of the method is 5.0 \u00d7 102 copies/50 \u00b5L per reaction [A lateral flow strip (LFS) is a very popular, conventional, portable, and rapid optical biosensor. To diagnose the rise in the incidence of reaction .SPR is an optoelectronic phenomenon where a ray passes through a specific prism and is collimated to a photodiode array detector at a definite refractive index. When the frequency of the incident radiation becomes equal to the resonance frequency of the metal, electrons from the metal surface move after gaining energy from the photon. This creates an electric wave called a plasmon. The SPR phenomenon is driven by the refractive index (RI) of the metal, and any binding event brings about a change in the RI of the metal surface, which in turn changes the reflection angle of the light. This class of biosensors provides fast, label-free real-time monitoring of analyte concentrations, with minuscule sample sizes and reusable sensor chips. These sensitive analyses can be performed in a short time with minimal reagents. This mechanism is tuneable with various materials like quantum dots, metallic nanoparticles, graphene, graphene oxide, nanocages, nanorods, spherical gold nanoparticles (AuNPs), nanocages, and nanorods .C. albicans was covalently immobilized on mixed self-assembled monolayers by 11-mercaptoundecanoic acid and 3-mercaptopropanol. Using SPR, the detection limit of direct detection of 107 cells/mL was increased to 106 cells/mL using the sandwich antibody. The specificity for C. albicans was noteworthy among other oral pathogens, viz., Escherichia coli, Staphylococcus aureus, Streptococcus mutans, \u03b2-streptococci, and Lactobacillus casei [C. albicans was fabricated. Briefly, silver nanospheres functionalized with monoclonal anti-Candida IgG antibodies were tethered on a glass slide to detect pathogenic antigen concentrations as low as 50 ng/mL. At this concentration, a red shift of at least 8nm was observed in the spectrum, and it increased to 25 nm at 200 ng/mL [A capture antibody against us casei . A local00 ng/mL .Candida monoclonal antibody-functionalized silver nanoparticles adhered on a glass slide. The binding of C. albicans antigens at very low concentrations of 50 ng/mL yielded an LSPR effect for nanosensing. Computational simulations, the spatial distribution of electromagnetic field enhancement around the nanoparticles, and molecular and bulk sensitivity were the parameters used for the analyses [An LSPR-based optical immunosensor platform for molecular biosensing has been engineered using anti-analyses .It is possible to upgrade SPR biosensors to miniature plasmonic nanobiosensors by modulating each chemical sample on the chip. Portability, real-time sample analyses, and the use of metamaterials are some of the issues of this category. Chemical stability, easy availability, lower costs, and thence commercialization may be achieved by replacing AuNps with TiNPs .Clinical diagnostics has taken a huge leap during the COVID-19 pandemic with reverse transcriptase PCR (RT-PCR) emerging as one of the primary tools due to its high sensitivity and low turnaround time. Other PCR-dependent tools are amplified fragment length polymorphism (AFLP), restriction fragment length polymorphism (RFLP), nested PCR, random amplification of polymorphic DNA (RAPD), multiplex PCR, and single-strand conformational polymorphism (SSCP) analysis .Candida species can be identified by PCR amplification of their nucleic acid material with specific primers or pan-fungal primers from any infected sample. In the case of the latter, the products can be sequenced to achieve correct species identification [Histoplasma capsulatum, causing serious pulmonary histoplasmosis, has been designed on the basis of three capture probes and three labeling oligonucleotide probes. Biotin-labeled probes were immobilized on streptavidin-coated 96-well plates [C. albicans culture showed a sensitivity of 10 CFU/mL and 0.2 CFU/mL, respectively [Candida isolates has been reliably and accurately performed using real-time PCR followed by high-resolution melting analysis. This closed-tube powerful tool can detect changes even up to single-nucleotide polymorphisms, mutations, epigenetic information, and species identification by the comparison of the shapes and relative positions of the melting curves. In this way, eight species can be easily identified: C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, C. krusei, C. lusitaniae, C. kefyr, and C. guillermondii [C dubliniensis, a predominant oral pathogen from cancer patients, could be identified along with C. albicans [C. albicans genomic DNA within 1 h [C. auris has been developed by Biopremier.The integration of PCR with other transducer modalities will hence lead to the development of point-of-care biosensors. fication ,66. An ul plates . A similectively ,68. The ermondii . Similaralbicans . The polthin 1 h . A real-Candida species, e.g., azole resistance in C. auris and caspofungin resistance in C. albicans, C. tropicalis, and C. parapsilosis [The discussion of nucleic acid biosensors seems to be incomplete without the mention of clustered regularly interspaced short palindromic repeat (CRISPR)-based biosensors. Despite its shortcomings like a costly thermocycler and the need for trained professional technicians, CRISPR is one of the powerful nucleic acid tools for discovering new therapeutic targets and pinpointing, understanding, and possibly avoiding the emerging drug resistance spectrum in psilosis . Dubey epsilosis . DiffereNanomaterials are game-changers in the field of biosensors. They have tuneable physicochemical and electrochemical properties and hence can be functionalized in several ways. They have a high surface-to-volume ratio, good biocompatibility, chemical stability, and miniaturization potential. Their electronic performance can be modulated by doping and modulating energy gap distributions. Due to their very close energy levels, an infinitesimal change in an energy level is promptly detected, leading to high sensitivity towards bioanalytes. Therefore, they can be tracked by optical , thermal (temperature change), physical (shape change in the presence of stress), and electronic means. The shape modulation of nanomaterials into nanoparticles, nanosheets, nanowires, nanotubes, nanocages, and nanoarrays is another advantage.Candia species like C. albicans, C. krusei, C. parapsilosis, and C. tropicalis. Characteristic impedimetric responses were obtained due to the selectivity of lectins, either concanavalin A or wheat germ agglutinin-modified gold nanoparticles on the cysteine monolayers, towards carbohydrate moieties on the yeast cell wall. Gold nanoparticles have good oxidative stability and a very low Dru. The charge transfer resistance was proportional to Candida CFU. The detection limit was within 102 to 106 CFU/mL, which was evaluated by electrochemical impedance spectroscopy and atomic force spectroscopy. The sensitivity of the ConA-based biosensor was C. parapsilosis < C. tropicalis < C. albicans < C. krusei, and that of the agglutinin-based biosensor was C. tropicalis < C. albicans < C. parapsilosis < C. krusei [S\u00e1 et al. have developed an innovative, simple, and sensitive lectin-based electrochemical impedance biosensor suitable for species identification of . krusei .C. albicans detection has been designed based on a single-walled carbon nanotube (SWCNT)-mediated electron transfer process [C. albicans, C. parapsilosis, C. krusei, C. tropicalis, and C. glabrata\u2014in a patient\u2019s blood. Nanoparticles tagged with complementary \u201ccapture probes\u201d were allowed to bind with amplified pathogenic DNA obtained after lysis. Nanoparticles with complementary \u201ccapture probes\u201d could then bind to the amplified DNA. Detection of up to 1\u20132 CFU/mL was performed with T2 magnetic resonance without any false positives [An immunosensor for  process . Neely eositives .C. auris genomic DNA at levels as low as 6 CFU/mL  compact shell layers prepared via ultrasound-induced polymerization attaches specifically to the fungal cell walls present in the complex samples within 10 s. Subsequently, fungal cells are identified by surface-enhanced Raman spectroscopy (SERS) within 10 min. NIPAM is thermoresponsive, AA is the coupling unit, and magnetic nanoparticles and caspofungin offer structural stability and specificity, respectively. Its sensitivity is 102 cells/mL. Identification is performed by determining differences in cell wall components of each strain, depicted as different peak values in the Raman spectrum [In an interesting development, a non-destructive organic\u2013inorganic hybrid nanocatcher has been used for the ultrafast capture of invasive spectrum .C. albicans. The genomic DNA of C. albicans was amplified at 64 \u00b0C for 40 min by a set of six primers which were designed based on C. albicans species-specific internal transcribed spacer genes. The results from LAMP were reported visually by LFB within 2 min with a sensitivity as high as 1 fg of genomic DNA. Since there were no cross-reactivities with the non-albicans strains, the analytical specificity was completely 100%. The whole procedure was completed within 85 min . The diagnostic accuracy was 100% [C. albicans targeting. The sensitivity is 200 fg of genomic DNA [A novel, easy, rapid, low-cost LAMP combined with nanoparticle-based lateral flow biosensor (LAMP-LFB) assay has been developed for was 100% . Anotheromic DNA .C. auris genosensor has been fabricated using ninhydrin as the DNA hybridization indicator by Guedes et al. Ninhydrin is an economical electrochemical DNA hybridization indicator with low oxidation potential and an environment-friendly molecule. It shows an increase in the oxidation peak during duplex formation. Twenty-five-base-long oligos were used as capture probes to locate C. auris genomic DNA in the urine samples. The detection level was 4.5 pg \u03bcL\u22121 [Recently, an electrochemical, highly reusable,  pg \u03bcL\u22121 .A multiplexed plasmonic optical nanosensor array has been designed for the detection of pathogenic fungal DNA sequences. Arrays of microspots of capture DNA sequence-functionalized gold nanoparticles bind with the target sequence at the functionalized spot. The capture sequences are complementary to the pathogenic analyte DNA. The resultant change in the local RI due to the binding event is detected as a peak shift in the local SPR spectrum. Non-specific binding was minimized by passivation with herring sperm DNA and coadsorption with mercaptohexanol. The sensor can be regenerated by dehybridization with 10 mM HCl .Candida pathogens like C. parapsilosis, C. auris, and C. albicans (Candida). Eluted bound aptamers are PCR amplified with phosphate-labeled primers and Cy5. After strand separation, followed by repeated (eight) rounds of target binding and amplification, these are subjected to analytical techniques of fluorescence microscopy, the fluorometric assay for fluorescent labeling, and flow cytometry [As a step towards the next generation of electronic biosensors, the polyclonal Systematic Evolution of Ligands by EXponential enrichment (SELEX) aptamer library has been utilized for the differential identification and fluorescence labeling of both non-albicans and albicans albicans . The sinytometry .Candida. Its biofilm-forming potential is aided by quorum-sensing molecules like tryptophol, aromatic alcohols, farnesol, tyrosol, and phenylethanol. Combining 10% each of MWCNTs and crown ether, 12-crown-4-ether, improved the selectivity and sensitivity of tryptophol detection to 1 \u03bcg/mL. This combination yielded the highest oxidation response due to only one electrochemically active phenolic active site of tryptophol [The ability to colonize various parts of the human body is another dangerous feature of yptophol .Aspergillus galactomannan analyses [Candia diagnosis.Recently, a nanobiosensor has been reported for analyses . Such stCandida sp. [Candida surveillance. PNA FISH probes hybridize with target DNA with high specificity, sensitivity, and strength, primarily due to their structure\u2019s electrical neutrality (absence of phosphate groups). Hybridization occurs rapidly (within a couple of hours) with low background noise. Faster species identification has been achieved with Yeast Traffic Light peptide nucleic acid FISH (PNA-FISH). Here, yellow light is generated for C. tropicalis, green light for C. albicans and C. parapsilosis, and red for C. krusei and C. glabrata [Candida species were rapidly identified from blood and peritoneal fluid cultures by PNA-FISH [Molecular diagnostics are novel and emerging biosensing methods that work on various microorganisms, including the detection of dida sp. ,78. The glabrata . In a clPNA-FISH .C. tropicalis cells which were permeabilized to internalize the hybridization probes [Silicon-based organic polymeric microfluidic hydrodynamic cell trapping has been integrated with PNA-FISH candiduria diagnostics. It offers direct target visualization amongst complex biological matrices with an epifluorescence microscope within a very short pre-enrichment time with a \u00b5L volume of samples. The FISH protocol was applied to trapped n probes .Candida biosensing. A POC device has five components\u2014a target, a probe, a sensing module, a transducer, and a signal read-out device. A microfluidics-based detection system has been applied for C. albicans in blood [Point-of-care (POC) devices are essential to reap the benefits of in blood . CombiniC. albicans infection in human blood [The internal transcribed spacer (ITS), a part of the ribosomal RNA gene complex, has been targeted for candidemia surveillance by quantitative PCR displaying a sensitivity of 0.2 CFU/\u00b5L. This region is present in 55 copies/haploid genome. It generates quantitative data to ease the evaluation of the magnitude of an blood .C. orthopsilosis, C. metapsilosis, C. parapsilosis, C. glabrata, and C. bracarensis species could not be identified by the biochemical approach but could be identified by MALDI-TOF MS systems [Another method, MALDI-TOF, is now being gauged as one of the diagnostic procedures for patient samples. MALDI-TOF mass signatures are used to identify the clinical fungal species as well as the associated drug resistance. The MALDI-TOF spectrum peaks are compared with signature peaks from reference spectra that are contained in a database. It is one of the fast, accurate, and straightforward methods of identification . In fact systems . HoweverCandida species was initiated as early as 1986 [6\u20135 \u00d7 108 cells cm\u22123. However, there was a gap in this direction until 2013, when gradually studies picked up. Further studies in this direction received a boost from 2021 onwards, perhaps coinciding with the emergence of COVID-19 detection systems , the need for sample preparation, and the need for regular and careful calibration .Candida infections, as traditional diagnostic methods are time-consuming and expensive and require skilled personnel, while biosensors offer rapid, accurate, real-time detection of Candida species. The use of biosensors can also reduce the cost of diagnosis and improve patient outcomes. Electrochemical biosensors are the most commonly used biosensors for the detection of Candida species. They are simple, cost-effective, and offer rapid detection. Optical biosensors are also promising for the detection of Candida species. They offer high sensitivity and specificity and can detect multiple analytes simultaneously.The use of biosensors offers a smart solution for rapid and accurate detection, with various biosensor platforms and emerging technologies that can improve point-of-care diagnosis being available. The use of biosensors can revolutionize the diagnosis of Candida species at very low concentrations. Emerging technologies that can improve point-of-care diagnosis for Candida infections include microfluidics, lab-on-a-chip, and smartphone-based biosensors. Microfluidics and lab-on-a-chip technologies offer miniaturized biosensors that can be used for point-of-care diagnosis. They are portable and easy to use and offer rapid detection. Smartphone-based biosensors are also promising for point-of-care diagnosis and offer a low-cost solution for rapid and accurate detection of Candida species.Nanobiosensors are emerging biosensors that offer high sensitivity and specificity, with the capability to detect Candida infections, being fast, accurate, and cost-effective. These technologies offer miniaturized biosensors that can be used for point-of-care diagnosis, proving portable and easy to use and offering rapid detection. The use of biosensors can revolutionize the diagnosis of Candida infections and improve patient outcomes. Further research is needed to optimize biosensor platforms and emerging technologies for the detection of Candida species.In conclusion, biosensors offer a smart solution for the diagnosis of"}
+{"text": "Sphagnum wetlands are global hotspots for carbon storage, conventionally attributed to the accumulation of decay-resistant litter. However, the buildup of mineral-associated organic carbon (MAOC) with relatively slow turnover has rarely been examined therein. Here, employing both large-scale comparisons across major terrestrial ecosystems and soil survey along Sphagnum gradients in distinct wetlands, we show that Sphagnum fosters a notable accumulation of metal-bound organic carbon (OC) via activating iron and aluminum (hydr)oxides in the soil. The unique phenolic and acidic metabolites of Sphagnum further strengthen metal-organic associations, leading to the dominance of metal-bound OC in soil MAOC. Importantly, in contrast with limited MAOC sequestration potentials elsewhere, MAOC increases linearly with soil OC accrual without signs of saturation in Sphagnum wetlands. These findings collectively demonstrate that Sphagnum acts as an efficient \u2018rust engineer\u2019 that largely boosts the rusty carbon sink in wetlands, potentially increasing long-term soil carbon sequestration. By employing large-scale comparisons across major terrestrial ecosystems and soil survey along Sphagnum gradients in distinct wetlands, Sphagnum is shown to act as an efficient rust engineer boosting the rusty carbon sink in wetlands Sphagnum is a well-known \u2018peat builder\u20194. More than half of northern wetlands (including peatlands) develop from Sphagnum-dominated landscapes, contributing to approximately 15% of soil carbon pool globally7. Cultivation and restoration of Sphagnum are advocated in many countries to recover ecosystem functions including carbon sequestration8. The standing paradigm considers that the tremendous carbon storage in Sphagnum-dominated wetlands (abbreviated as \u2018Sphagnum wetlands\u2019 hereafter) is mainly attributed to the inhibited microbial decomposition as well as low decomposability of the recalcitrant Sphagnum litter11, leading to soil organic carbon (SOC) accumulation mainly as poorly degraded plant detritus or particulate organic carbon (POC)12. By comparison, organic carbon (OC) associated with minerals  is a more persistent SOC pool with longer turnover times than POC14, since the accessibility of mineral-associated organic carbon (MAOC) to decomposers is limited15. However, the buildup of MAOC has rarely been investigated in Sphagnum wetlands, which may underpin long-term SOC accrual and dynamics under global changes.17. In particular, phenolic metabolites of Sphagnum (such as sphagnum acid) may induce the transformation of pedogenic iron (Fe) (hydr)oxides to reactive  species and promote strong mineral protection of SOC18. Sphagnum-induced acidic and water-saturated environments9 may also facilitate mineral weathering and enhance the production of SRO Fe and aluminum (Al) (hydr)oxides20, which can effectively bind OC23 and potentially contribute to MAOC accrual. More importantly, Sphagnum-induced transformation and production of SRO Fe and Al (hydr)oxides may increase the sequestration capacity of MAOC, which is shown to saturate in upland  soils due to limited availability of reactive minerals 25. However, metal-bound OC (abbreviated as \u201cbound OC\u201d hereafter) is rarely examined in Sphagnum wetlands, and its contribution to SOC or MAOC has not been assessed in comparison to other terrestrial ecosystems26. Hence, it is essential to clarify whether Sphagnum has a prevalent enhancement impact on metal-organic associations at the landscape scale and whether Sphagnum may elevate MAOC sequestration potentials via activating metal oxides. Answering these questions may not only reveal an underappreciated mechanism for SOC sequestration in Sphagnum wetlands, but also aid in the effort to increase the saturation level of MAOC to stabilize soil carbon in terrestrial ecosystems24.Emergent evidence suggests that MAOC constitutes a significant fraction of SOC in wetlands20, which show contrasting distributions in different geological settings, e.g., igneous vs. sedimentary rocks. Vast areas of wetlands worldwide are located in volcanic and tephra-receiving areas27, which facilitate Sphagnum establishment29. Soils in such areas  are replenished with Fe and Al minerals31, resulting from parent material weathering and eolian deposition of volcanic glass32. Alternatively, Sphagnum-dominated wetlands are also commonly found in sedimentary landscapes such as karst regions that are relatively deprived of SRO Fe and Al (hydr)oxides but rich in less weatherable phyllosilicates33. There is little information, however, on how Sphagnum\u2019s enhancement of metal-organic associations (if present) varies across these different geological settings.Reactive Fe and Al (hydr)oxides in soils are ultimately derived from Fe- or Al-bearing minerals in parent materialsSphagnum\u2019s control on mineral protection of SOC by metal oxides. We first compared the contents of bound OC and reactive metals in the surface soils (0\u201325\u2009cm) of Sphagnum wetlands with non-Sphagnum wetlands and other major terrestrial ecosystems by compiling published data . Wetlands were further divided into Sphagnum and non-Sphagnum wetlands.Using compiled published data and a national survey of wetlands in China, we generated a dataset of 512 measurements of bound OC in the surface soils (0\u201325\u2009cm) of diverse terrestrial ecosystems globally , followed by non-Sphagnum wetlands  and permafrost soils  based on one-way ANOVA , in comparison with either non-Sphagnum wetlands  or the average value of all terrestrial ecosystems estimated by ref.\u200926 which barely included Sphagnum-influenced landscapes 34, the highest reactivity of Fe (hydr)oxides indicated by the ratio of Feo to dithionite-extractable Fe35 34, and the lowest clay content among all terrestrial ecosystems  and mean annual temperature (MAT) as non-Sphagnum wetlands, which were comparable to some other examined ecosystems  as forests in our dataset , molar ratio of bound OC to weight-normalized contents of Fed and Ald  as a proxy for metal-organic association strength37, and sulfate-extractable Ca 38. Compared with non-Sphagnum wetlands, Sphagnum wetlands had higher molar ratio of bound OC:(0.5Fed\u2009+\u2009Ald), Feo:Fetotal and Alo:Altotal, suggesting a stronger metal-organic association and a higher proportion of reactive species in total Fe and Al 37 in our surveyed wetlands , followed by water-soluble phenols  and soil moisture content . Soil pH negatively affected bound OC , while other soil properties (such as clay\u2009+\u2009silt and Cas) had no influence. These results suggested that Sphagnum induced strong metal-organic associations primarily by activating Fe and Al (hydr)oxides in the soil, which was likely further strengthened by increasing phenolic metabolites, acidity and moisture18. These variables were, however, correlated in the field, and their separate influences on metal-organic associations need to be confirmed with future experimental approaches.To ascertain the main drivers leading to the storage of bound OC in nds Fig.\u00a0. A princnds Fig.\u00a0 and, corSphagnum\u2019s effect on metal-organic associations was further investigated with both spatial and temporal gradients of Sphagnum in four distinct wetlands. The spatial gradient included natural vegetation successions in wetlands with varied coverage of Sphagnum from 0 to 100%, representing a gradual shift from Carex to Sphagnum as the dominant species . The temporal gradient was based on a cultivation project in Dushan, involving varying cultivation time of Sphagnum (0\u201320 years) in rice paddy soils. Sphagnum expansion (reflected in an increasing coverage or cultivation time) significantly increased SOC contents (by 11\u201320%), the proportion of bound OC in SOC (by 5\u201316%) as well as SRO Fe and Al  at two surface depths (0\u201310 and 10\u201320\u2009cm) of all sites  also increased at a similar magnitude among sites oxides and organic matter37  outpaced that of Alo:Altotal  may reductively dissolve ferric Fe to ferrous Fe oxides was accompanied (and likely facilitated) by Sphagnum-induced acidity and water saturation  Al (hydr)oxides.To complement the large-scale comparison across terrestrial ecosystems, Sphagnum primarily enhanced metal-organic associations by activating soil Fe and Al (hydr)oxides, we further tested whether Sphagnum\u2019s effect varied in geological landscapes with different contents of transformable Fe and Al minerals. We selected a subset of 12 Sphagnum wetlands among our surveyed sites , and paired them with the non-Sphagnum wetlands in the same area. A response ratio (RR) of bound OC and related variables was calculated, i.e., the natural logarithm-transformed ratio of a specific variable in the Sphagnum wetland relative to that in the paired non-Sphagnum wetland, to reflect Sphagnum\u2019s influence under similar geological and climatic settings. A positive value of RR indicated an enhancement of the examined variable in the Sphagnum than non-Sphagnum wetlands, and vice versa. Given different variances of the calculated RR for different sites, the weight of each site was estimated based on the reciprocal of the variance for individual RRs. We further evaluated site-weighted response (RR++) by weighting the RR of six individual sites with the inverse variance (see details in Methods) for the igneous and sedimentary rock-based wetlands, respectively.As ++) in soil pH and Al reactivity in response to Sphagnum dominance in the igneous and sedimentary rock-based wetlands, the increase of bound OC was larger in the igneous than sedimentary rock-based wetlands  species therein. Additionally, bound OC was more responsive to Feo change in the igneous rock-based wetlands oxides may be a hotspot for Sphagnum\u2019s enhancement on metal-organic associations.Despite similar magnitudes of change  and evenly distributed along the 1:1 line in all our surveyed Sphagnum-influenced wetlands, while bound OC had overall much lower contents than MAOC in non-Sphagnum wetlands  encompassed a much wider range and higher values of both SOC and MAOC contents (per gram of soil) than uplands . However, MAOC increased linearly with SOC in the Sphagnum-influenced wetlands within our examined range (up to 400\u2009mg SOC g\u20131 soil), so that saturation of MAOC was not observed. This result suggested that Sphagnum largely increased the sequestration capacity of MAOC via promoting the formation of reactive Fe and Al (hydr)oxides in wetlands.Given the paucity of published MAOC data in wetlands, we further examined the MAOC\u2013SOC relationship in our studied wetlands in comparison with published results from uplands including forests and grasslandsnds Fig.\u00a0. Similarnds Fig.\u00a0, suggestSphagnum in distinct wetlands oxides to protect SOC  induces the concentration of reactive metal oxides and bound OC in the soil. Moreover, Sphagnum may enhance metal-organic association strength oxides and organic matter via complexation and/or co-precipitation 45 under microbial inhibition11, our study reveals an underappreciated mechanism, i.e., mineral protection of SOC by metal oxides, that contributes to a remarkable accumulation of MAOC in Sphagnum wetlands  of total SOC stock under Sphagnum dominance  than POC14, Sphagnum-induced MAOC accumulation may underpin wetland SOC stabilization in the long term and deserve more attention especially under ongoing climate changes inducing water-table fluctuations16 and Sphagnum distribution shifts47.First, while a large number of studies have mainly attributed the tremendous carbon storage under nds Fig.\u00a0. Admittence Fig.\u00a0. TherefoSphagnum wetlands of this study), it is widely observed that MAOC accumulation plateaus during SOC accrual oxides in the soil and greatly increases the sequestration capacity of MAOC . However, based on the project in Dushan, we estimate that Sphagnum cultivation increased MAOC at 1.58\u2009Mg C ha\u20131 yr\u20131 in the surface soils (0\u201320\u2009cm), accompanied by ~60% increase of SRO Fe and Al (hydr)oxides in a relatively short period 50 and forests (up to 0.008\u2009Mg\u2009C\u2009ha\u20131\u2009yr\u20131)51 during vegetation restoration projects in China, suggesting a sizable sequestration potential of MAOC alone following Sphagnum restoration in wetlands. Hence, Sphagnum restoration and cultivation may provide a promising nature-based solution to enhance MAOC sequestration and SOC persistence.Second, in upland soils  characterized by thick peat layers52. In the peat layers that are mostly plant residuals with very low mineral contents, Sphagnum may have a limited effect on metal-organic associations. Nevertheless, high Fe content has been reported for the peat layers in boreal Sphagnum wetlands55, and \u2018bog iron\u2019, a reactive Fe-rich layer, is often found close to the soil surface underlying peat layers due to precipitation of Fe57. Hence, the applicability of our findings to peat layers and the underlying mineral soils in those peat-rich Sphagnum wetlands deserves future study. Moreover, while our study focuses on wetlands, the utility of Sphagnum cultivation in less saturated ecosystems (such as moist forest) for carbon sequestration also deserves investigation. These considerations may constrain the feasibility and uncertainties associated with Sphagnum cultivation for SOC sequestration. Nevertheless, our study highlights that unique interactions between plants (Sphagnum) and geology  may provide an overlooked solution to advance our ongoing efforts to manage carbon sequestration for climate change mitigation.Admittedly, MAOC sequestration potential under Sphagnum wetlands with other major terrestrial ecosystems, we compiled related data from the National Ecological Observatory Network (NEON) database58. We supplemented the database by searching on the Web of Science and Google Scholar with the following keywords: \u201cwetland\u201d OR \u201cpeatland\u201d OR \u201cbog\u201d OR \u201cfen\u201d OR \u201cmarsh\u201d OR \u201cswamp\u201d OR \u201cpermafrost\u201d OR \u201cforest\u201d OR \u201cgrassland\u201d AND \u201cmetal-organic association\u201d OR \u201cbound OC\u201d OR \u201creactive metal\u201d OR \u201coxalate-extractable Fe and Al\u201d OR \u201cdithionite-extractable Fe and Al\u201d. Only references using the same extraction protocols for bound OC, i.e., by the citrate-bicarbonate-dithionite (CBD) method21, and reactive Fe and Al species  were included. We compiled a final dataset containing 272, 650, 520 and 714 measurements of bound OC, Feo, Alo, and Fed, respectively (Supplementary Data\u00a0http://data.isric.org) were used to obtain related information.To compare contents of bound OC and reactive Fe and Al (hydr)oxides in the surface soils (0\u201325\u2009cm) of Sphagnum wetlands, we supplemented the global dataset by surveying 20 Sphagnum wetlands and 29 non-Sphagnum wetlands with diverse climatic, geological and soil characteristics across China between 2019 and 2022 . The distance between the Sphagnum and paired non-Sphagnum wetlands varied between 500\u2009m and 2\u2009km. It should also be mentioned that the majority of our surveyed Sphagnum wetlands (14 out of 20) were minerotrophic, while the rest were ombrotrophic. Most of our surveyed Sphagnum wetlands also had a thin layer of peat  in Jinchuan, Hani and Dajiuhu wetlands. Both Jinchuan  and Hani wetlands  are located on the flank of Changbai Mountains, Northeast China, developed from basalt platforms along the Longgang Volcanic field59 with the upland soils classified as Histic Andosols . MAT is 3.3 and 4\u2009\u00b0C, and MAP is 1054 and 750\u2009mm for Jinchuan and Hani, respectively. The dominant plant species are Sphagnum palustre, Carex schmidtii, Carex lasiocarpa and Polytrichum strictum, etc. in Jinchuan, and Sphagnum palustre, Sphagnum magellanicum and Carex lasiocarpa in Hani. Dajiuhu wetland  is located in a subalpine basin (1700\u2009m a. s. l.) of Mt. Shen Nong Jia, Hubei Province, surrounded by steep mountains developed from igneous and carbonate rocks60. It was formed by the combined effects of glaciers and karstification in the Late Pleistocene. The region has an MAT of 7.2\u2009\u00b0C and MAP of 1560\u2009mm, with upland soils classified as Histosols . The dominant plant species are Sphagnum palustre, Carex argyi, Juncus effuses and Sanguisorba officinalis. All three wetlands were sampled in July 2020 or 2021. Four Sphagnum coverage gradients were selected at each location , representing a gradual shift from Carex to Sphagnum as the dominant species.To further track the dynamic change of metal-organic associations with num Fig.\u00a0. The spaSphagnum cultivation was selected in Dushan , Guizhou Province, one of the largest Sphagnum cultivation bases in Southwest China61. The region has an MAT of 15\u2009\u00b0C, MAP of 1430\u2009mm, and widespread Sphagnum wetlands developed on karst landforms with soils classified as Histosols . Historically, pristine Sphagnum wetlands were drained for agriculture (mainly rice paddies). In the past 20\u201330 years, Sphagnum has been cultivated for restoration and business purposes, producing temporal gradients from 0 to 20 years of Sphagnum cultivation  for soil sampling. For comparison, we also collected soils from pristine Sphagnum wetlands and rice paddies managed for >60 years nearby .A temporal gradient of ion Fig.\u00a0. The SphSphagnum expansion gradients in Jinchuan, Hani, Dajiuhu and Dushan, mineral soils were sampling using PVC pipes  with both ends sealed by plastic film. The collected soil columns were further divided into two subsamples (0\u201310 and 10\u201320\u2009cm). For all samples, a small aliquot of the soil was immediately mixed in 0.5-M hydrochloric acid (HCl) to extract ferrous iron [Fe(II)] (described below) and saved for moisture determination, while the rest was freeze-dried and sieved (<1\u2009mm) with roots removed by hand before further analysis.At each sampling site, litter or peat layer was collected in a quadrat (20\u2009cm\u2009\u00d7\u200920\u2009cm) with the layer depth measured. The underneath mineral soil was sampled down to 20\u2009cm with PVC pipes  and transported (cooled by ice bags) to the laboratory immediately  and acetanilide. The accuracy  and precision  for the OC analysis were \u00b10.21% and 0.28% determined by the soil standard, respectively (n\u2009=\u20096), and were \u00b10.14% and 0.17% determined by acetanilide, respectively (n\u2009=\u20096). Organic carbon stocks (g\u2009cm\u20132) in the litter layer or surface soils (0\u201320\u2009cm) were calculated as:\u20131), bulk density (g\u2009cm\u20133) and depth (cm) of litter and surface mineral soils, respectively.The field moisture content and bulk density of soils were determined by thermogravimetric desiccation at 105\u2009\u00b0C for 24\u2009h. Soil pH was measured at a soil:water ratio of 1:5 (w:v). The OC contents of bulk soils and litter were analyzed by an elemental analyzer  after fumigation by concentrated HCl for 96\u2009h to remove carbonates (no inorganic carbon was detected in fumigated soils)63 at 750\u2009nm by a Multi-Mode Microplate Reader . Total contents of major metals in bulk soils, including Fe, Al and manganese (Mn) were analyzed using an X-ray fluorescence analyzer  and denoted with a subscript \u2018total\u2019. Soil samples were prepared as pressed pellets (40-mm diameter) with a semi-automatic pressor . Contents of the examined metals were calibrated against calibration models created with 19 certified reference soil materials (GSS1-GSS19) from the National Research Centre of China. The accuracy and precision of the applied method were checked by measurements of certified reference GSS2, GSS5 and GSS7. The accuracy (RPD%) for total Fe, Al and Mn analysis was \u00b11.5%, \u00b11.2% and \u00b12.2%, respectively (n\u2009=\u20099). The averaged precision (RSD%) for total Fe, Al and Mn analysis was 1.8%, 1.4% and 2.9%, respectively, based on three certified materials.Water-soluble phenols were extracted by mixing 20\u2009mL of Milli-Q water with 1\u2009g of freeze-dried soil for 2\u2009h, followed by centrifugation and filtration (0.45\u2009\u03bcm), and determined using the Folin-Ciocaltu method64, dried soils were treated repeatedly with hydrogen peroxide solution (10%) to remove organic matter until no bubbles were produced, and then boiled with HCl (10%) to remove lime. The residues were repeatedly rinsed with deionized water and then dispersed in sodium hexametaphosphate by sonication. Soil texture was measured using a laser diffraction using a Malvern Mastersizer 2000 particle analyzer  with particles <2\u2009\u00b5m and of 2\u201350\u2009\u00b5m defined as clay and silt, respectively.For soil texture (clay and silt) measurementHCl] was isolated from 1\u2009g of fresh soil using 5\u2009mL of 0.5-M HCl immediately upon sampling. After centrifugation, an aliquot of the supernatant reacted with 5-mM ferrozine solution, and Fe(II) was measured using the ferrozine-ultraviolet absorbance method65 at 562\u2009nm on a UV-Vis spectrometer . A standardized calibration curve of ferrous ammonium sulfate (0\u201350\u2009mg\u2009L\u20131) was made using the same procedure described above.To probe Fe transformation and speciation in wetlands, acid-extractable ferrous iron [Fe(II)67, were examined using a selective dissolution procedure involving extraction by ammonium oxalate-oxalic acid34 and the CBD method21, respectively. Feo and Alo represent poorly crystalline or SRO Fe and Al (hydr)oxides, while Fed and Ald represent SRO and crystalline Fe and Al not bound in silicates34. The suspensions were filtered through 0.45-\u03bcm filters, and dissolved metals were quantified on an inductively coupled plasma-optical emission spectrometer . Calibration solutions were prepared by dilution of certified standard solutions of Fe and Al (1000\u2009\u03bcg\u2009mL\u20131). The blank control was assessed by deionized water. The determinations were performed in triplicate to guarantee quality control procedures. The accuracy (RPD%) for Fe and Al were \u00b11.3% and \u00b11.1%, respectively. The precision (RSD%) for Fe and Al were 1.5% and 1.2%, respectively (n\u2009=\u20099). When assessing the role of reactive Fe and Al as a whole, we summarized the weight-normalized contents of Fe and Al as \u201c0.5Fe\u2009+\u2009Al\u201d to normalize the atomic mass difference between Fe and Al for graphing and statistical purposes37. To further explore the potential effect of Ca bridging on bound OC, we analyzed extractable Ca with sodium sulfate (Cas)38. Mn is also an important redox-active metal in soils. However, both total Mn and reactive  Mn were two orders of magnitude lower in the soil compared with Fe and Al in Sphagnum wetlands oxides, which are recognized important players in SOC stabilization21 was used to extract Fed and Ald from soils and to release OC bound to reactive metals. Dry soils (0.25\u2009g) were reacted with 15\u2009mL of CBD buffer solution  at 80\u2009\u00b0C in a water bath for 15\u2009min twice. After cooling, the supernatant was separated after centrifugation at 2100\u2009\u00d7\u2009g for 10\u2009min, and the CBD-treated soil residues were rinsed with sodium chloride  solution four times. An aliquot of dry soil (0.25\u2009g) was also extracted with NaCl (0.25\u2009M) in tandem as a control with another buffer solution (containing 1.6\u2009M NaCl and 0.11\u2009M sodium bicarbonate). The OC contents of soil residues after extraction by NaCl (OCNaCl) and CBD (OCCBD) were determined after freeze-drying. Bound OC (in percentage) was determined as:A modified CBD method68. Therefore, we calculated the molar ratio as:Al represent the molar mass of carbon and Al, respectively.The molar ratio of bound OC to metal oxides can indicate the association strength between metal oxides and organic matter, with lower values (<1) indicating sorptive interactions and higher values (>1) indicating complexation or co-precipitationSphagnum expansion, soils in Jinchuan, Hani, Dajiuhu and Dushan were physically separated into three fractions41. Briefly, dried and sieved soils were shaken in sodium polytungstate (SPT) solutions (1.6\u2009g\u2009cm\u20133) at a soil:solution ratio of 1:10 (w:v) for 30\u2009min at 180\u2009rpm to gently disperse aggregates, and subsequently centrifuged at 2800\u2009\u00d7\u2009g for 15\u2009min to isolate light fraction. The light fraction and high-density soil residues were washed with Milli-Q water until the solution conductivity was <50\u2009\u03bcS\u2009cm\u20133. The high-density residues were then wet sieved to obtain particulate organic matter  and mineral-associated organic matter . All three fractions were freeze-dried, weighed and ground to <0.25\u2009mm in an agate mortar. The OC content of each fraction was analyzed as described previously.To differentiate carbon accumulation in various pools of SOC under 69. Briefly, dried and sieved soils were wet sieved through 53\u2009\u03bcm. Size fractions of <53\u2009\u03bcm were further separated by density using SPT solutions (1.6\u2009g\u2009cm\u20133), removing light fraction and POM from MAOM. The resulting fraction (MAOM\u2009<\u200953\u2009\u03bcm) was repeatedly rinsed with Milli-Q water until the electric conductivity was <50\u2009\u03bcS\u2009cm\u20133. Samples were subsequently collected by centrifugation, freeze-dried, weighed and ground to <0.25\u2009mm in an agate mortar. The OC content of MAOM was analyzed as described previously.To compare bound OC and MAOC contents, we further separated MAOM from the remaining soils of our surveyed 49 wetlands according to ref.\u2009Sphagnum coverage and sites on the investigated parameters along the Sphagnum expansion gradient, followed by one-way ANOVA to test the effect of Sphagnum coverage or cultivation time for a specific site. Relationships between the tested variables were explored using Spearman correlation. PCA was conducted to examine the variance of bound OC with its potential influencing factors across different wetlands. Differences and correlations were considered to be significant at a level of p\u2009<\u20090.05.Data analysis was performed using the R software . Difference between groups was analyzed by one-way ANOVA. Two-way ANOVA was used to examine the effects of Sphagnum\u2019s influence, i.e., in Sphagnum relative to the paired non-Sphagnum wetland, RR was calculated for individual site70:Xt and Xc are the reported means for each soil variable in the Sphagnum and paired non-Sphagnum wetlands for each site, respectively. The variance (v) of the logarithmic effect size was calculated as follows:c and St are the standard deviations of Xt and Xc, and Nc and Nt are the sample sizes of data. The weighting function was calculated based on the reciprocal of the variance in individual RRs:To compare soil\u2019s responses to ++) for the igneous- and sedimentary rock-based wetlands, respectively. The weighted RR (RR++) was calculated from the individual RRij  by pairwise comparison between the Sphagnum and paired non-Sphagnum wetlands, where m is the number of groups and k is the number of comparisons in the ith group:We further consolidated the RR of individual site to evaluate site-weighted response (RR++ was estimated as follows:The standard error (S) of RR++ \u00b1 1.96\u2009S(RR++). The difference was considered to be significant, if the 95% confidence interval did not overlap between the igneous and sedimentary rock-based wetlands.The 95% confidence interval (95% CI) was RRSupplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data1Supplementary Data2"}
+{"text": "Phytophthora infestans, a highly destructive plant oomycete pathogen, is responsible for causing late blight in potatoes worldwide. To successfully infect host cells and evade immunity, P. infestans secretes various effectors into host cells and exclusively targets the host nucleus. However, the precise mechanisms by which these effectors manipulate host gene expression and reprogram defenses remain poorly understood. In this study, we focused on a nuclear-targeted effector, Pi07586, which has been implicated in immune suppression. Quantitative real-time PCR (qRT-PCR) analysis showed Pi07586 was significant up-regulation during the early stages of infection. Agrobacterium-induced transient expression revealed that Pi07586 localized in the nucleus of leaf cells. Overexpression of Pi07586 resulted in increased leaf colonization by P. infestans. RNA-seq analysis revealed that Pi07586 effectively suppressed the expression of PR-1C-like and photosynthetic antenna protein genes. Furthermore, high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS) analysis indicated that Pi07586 overexpression led to a substantial decrease in abscisic acid (ABA), jasmonic acid (JA), and jasmonoyl-isoleucine (JA-Ile) levels, while not affecting salicylic acid (SA) and indole-3-acetic acid (IAA) production. These findings shed new light on the modulation of plant immunity by Pi07586 and enhance our understanding of the intricate relationship between P. infestans and host plants. However billion . The ongsistance . The firsistance .Phytophthora pathogens secrete two groups of effectors (apoplastic and cytoplasmic effectors) into plant cells to facilitate infection by suppressing host defense and physiological processes  as a conserved hypothetical virulence effector gene was first reported in P. infestans indicates that Pi07586 was up-regulated during the early stages of infection , jasmonic acid (JA), and jasmonoyl-isoleucine (JA-Ile). Collectively, our research sheds new light on the perturbation of host immune responses at the transcriptome level by the oomycete effector Pi07586, filling the existing gap in nuclear effector-related investigations.Here, we identify and characterize that an effector Pi07586 localizes in the nucleus, and promotes 22.1Nicotiana benthamiana seeds, and the transient expression vector pRI101-GFP were provided by the Key Laboratory for Potato Biology of Yunnan province. Plants were grown under standard conditions  in the laboratory greenhouse. D\u00e9sir\u00e9e and the Pi07586 transgenic lines grow for ~5\u20136 weeks for P. infestans inoculation. The N. benthamiana grows ~4\u20135 weeks for transient expression. The Agrobacterium strain used for transient expression is GV3101 . The P. infestans strain 88069 was propagated and stored in the rye V8 medium, and incubated at 18\u00b0C for 14 days.Potato sterile seedlings of cultivar D\u00e9sir\u00e9e, 2.2P. infestans strain 88069 grown for 14 days was taken and ddH2O was added to the culture dish, with the sporangia being scraped off the dish with an applicator. After filtration with microcloth, the final concentration of suspension was adjusted to 50 sporangia/\u03bcL. The suspension was stored at 4\u00b0C for 1\u00a0h. Then 10 \u03bcL suspension was placed on both sides of the detached potato leaf, using ddH2O as the control. After 24\u00a0h of dark humid cultivation under 16\u00a0h light/8\u00a0h dark conditions, the potato leaves were taken at 0, 24, 48, and 72\u00a0h post infection and stored at -80\u00b0C for later experiments. Three samples at each time point were repeated for a total of three biological replicates.The 2.3Pief2 (XM_002901697.1) gene of P. infestans was used as the internal reference gene. The 2-\u0394\u0394Ct method was used to calculate the relative gene expression  as per the manufacturer\u2019s instructions. Reverse transcription was performed using the TaRaKa reagent  to obtain cDNA. The qRT-PCR reaction was conducted according to TB Green Premix Ex Taq II . The pression . The qRT2.4Agrobacterium tumefaciens carrying the p19, GFP-EV (pRI101-GFP), and GFP-Pi07586 (pRI101-GFP-Pi07586) recombinant plasmids were activated and cultured on LB solid medium with corresponding antibiotics at 28 \u00b0C for 2-3 days. The culture was then centrifuged at 3000 rpm for 10\u00a0min. A mixture of MMA  was prepared to achieve an OD600 of 0.01. GFP-EV and GFP-Pi07586 were combined with p19 in equal volumes and incubated for 1\u00a0h. The bacterial solution was injected onto the surface of N. benthamiana leaves using a needleless 1 mL syringe. After 36-48\u00a0h of transient expression, the epidermis was carefully removed using tweezers and placed on a glass slide. Observation and photography were conducted using a laser confocal microscope.2.5N. benthamiana plants were infiltrated with A. tumefaciens (OD600\u2009=\u20090.3) containing the targeted genes. Leaves were harvested 48\u00a0h post-infiltration (hpi) and 100 mg of leaf tissue was placed in 1 mL of PBS lysis buffer with protease inhibitors. After grinding and centrifugation, the sample concentration was confirmed using the BCA protein content detection kit . The protein sample was mixed with 1\u00d7 SDS-PAGE loading buffer and boiled to denature the protein. The processed sample (20 \u03bcL) was loaded onto an SDS-PAGE gel  and electrophoretically separated in 1\u00d7 MOPS SDS running buffer at 120\u00a0V for 1.5\u00a0h. One gel was transferred to a PVDF membrane at 240 mA for 1\u00a0h, while another gel was stained with Coomassie Blue Staining Solution  for loading visualization. The PVDF membrane was blocked with 5% milk in 1\u00d7 TBST for 1\u00a0h to prevent non-specific binding. The primary antibody  was diluted according to the instructions and incubated with the membrane. The membrane was washed thrice every 30\u00a0min with 1\u00d7 TBST. Subsequently, the PVDF membrane was incubated with the secondary antibody  at the recommended dilution in 1\u00d7 TBST. The membrane was washed thrice every 10\u00a0min with 1\u00d7 TBST. The Immobilion Western Chemiluminescence HRP Substrate color development kit  was added to the PVDF membrane, and a chemiluminescence instrument was used for luminescence photography.2.6Pi07586 overexpression vector was successfully constructed and transformed into Agrobacterium GV3101. Stem segments of the potato cv. D\u00e9sir\u00e9e were used for genetic transformation. Explants were infected with Agrobacterium and cultured in the Z1N2AS medium (MS20 1L+ZT 1 mL+NAA (10 mg/mL) 200 \u03bcL+AS 1 mL) for two days in darkness. Subsequently, the explants were transferred to the recovery medium (MS20 1L+ZT 2 mL+NAA (0.1 mg/mL) 100 \u03bcL+TMT 2 mL) where they were dedifferentiated and formed calli. The calli were then transferred to the differentiation medium Z2N0.01 (MS20 1L+ZT 2mL+NAA (0.1 mg/mL) 100 \u03bcL+TMT 2 mL+Kana 2 mL) and cultivated in a light incubator. After approximately two months, the calli differentiated into buds, and regenerated seedlings were obtained. Once the regenerated seedlings reached 1-2\u00a0cm in size, they were cut from the callus and transferred to the rooting medium (MS30 1L+TMT 2 mL+Kana 1 mL) for initial screening. Positive selection of Pi07586 was determined using PCR. The culture medium formulas mentioned above are prepared in a 1 L ratio.The method was performed as described previously . The pRI2.7Total RNA quality was assessed using the NanoDrop 2000 spectrophotometer and Agent2100/LabChip GX. Upon qualification, the library was constructed. Firstly, mRNA was enriched using magnetic beads containing Oligo (dT) and randomly fragmented using the Fragmentation Buffer. Then, the first and second cDNA strands were synthesized using mRNA as a template, followed by their purification. The purified double-stranded cDNA was subjected to end repair, A-tail addition, and sequencing connections. AMPure XP beads were used for fragment size selection. Finally, PCR enrichment generated a cDNA library. Following cDNA library quality inspection, the Illumina NovaSeq6000 sequencing platform was used for PE150 mode sequencing.2.8The sample was ground in liquid nitrogen until fully crushed, then accurately weighed and placed in a glass test tube. Then, 10 times the volume of acetonitrile solution was added along with 8 \u03bcL internal standard mother liquor. Overnight extraction was performed at 4\u00b0C, followed by centrifugation at 12000\u00a0g for 5\u00a0min, and the final supernatant was obtained. Subsequently, the extraction was repeated twice with the precipitate using 5 times the volume of acetonitrile solution, and then the resulting supernatants were combined. To this, 35 mg of C18 filler was added and shaken vigorously for 30 s, centrifuged at 10000\u00a0g for 5\u00a0min, and the supernatant was discarded. The residue was dried with nitrogen at a 400 \u03bcL methanol resolution rate, then passed through a 0.22 \u03bcM organic phase filter membrane, and finally stored in a -20\u00b0C refrigerator for machine testing. The hormone content was measured using HPLC-MS.33.1P. infestans identified a candidate virulence effector gene, Pi07586, with a length of 447 bp, encoding a 148-amino acid protein lacking an RxLR/LxLFLAK domain but containing an NLS motif  post-infestation, with water-treated leaves as controls. qRT-PCR analysis , we observed larger disease lesions on the leaves of Pi07586 transgenic plants compared to D\u00e9sir\u00e9e (P. infestans infection.To investigate the virulence function of Pi07586 during 3.3S. tuberosum DM reference genome (v6.1) (2(FC) was Soltu.DM.04G006870.2, encoding a transcription factor jumonji (jmj) family protein in the wild-type and transgenic plant comparisons. The second largest log2(FC) belonged to Soltu.DM.03G025990.1, which encodes a P-loop containing nucleoside triphosphate hydrolases superfamily protein. Notably, the downregulated gene with the largest fold change was a newly identified gene.Furthermore, we investigated the mechanisms of Pi07586 in potatoes. RNA-seq analysis was conducted on three biological replicates of D\u00e9sir\u00e9e, lines 2, and 10, 24\u00a0h of inoculation with the pathogen, with a total of nine samples The Illumina NovaSeq6000 sequencing platform was utilized for PE150 pattern sequencing. A total of 56.06 Gb of Clean Data was obtained, with each sample yielding 5.72 Gb of Clean Data, and a Q30 base percentage exceeding 91.70% (e (v6.1)  using DEe (v6.1)  for diff3.4Pi07586, we conducted GO to examine the transcriptome changes. GO analysis encompasses three main branches: biological process, molecular function, and cellular component. Subsequently, we assigned DEGs to specific functional terms by annotating and classifying them at the GO database\u2019s secondary classification level, thus providing insights into their primary functions. To understand the primary biological functions of the DEGs triggered by In contrast, GO enrichment analysis of downregulated DEGs reveals enrichment in photosynthetic-related processes, cellular components, and molecular functions Table\u00a02.3.5To comprehend the biochemical metabolic and signal transduction pathways linked to DEGs, we performed KEGG annotation and functional classification analysis on 793 DEGs obtained from wild-type and transgenic lines. DEG annotation results were categorized into five types of KEGG pathways: cellular processes, environmental information processing, genetic information processing, metabolism, and organismal systems  and photosynthetic antenna protein  showed a significant decrease compared to D\u00e9sir\u00e9e  for both D\u00e9sir\u00e9e and transgenic 2 and 10 lines. Interestingly, transgenic lines showed reduced ABA, JA, and JA-Ile compared to D\u00e9sir\u00e9e Figure\u00a064P. infestans successfully causes late blight on tomatoes and potatoes, which is inseparable from a large number of apoplastic and cytoplasmic effectors. Apoplastic effectors accumulate in the intercellular spaces and interact with extracellular defense-related factors, whereas cytoplasmic effectors are secreted into intercellular spaces of host cells and are recognized by host plants to activate immunity or act as a virulence factor to suppress plant immunity  plays a vital role in plant developmental processes and immune responses to pathogen attacks. Previous research has shown that ethylene response factors (ERFs) can positively or negatively regulate plant resistance. For instance, AtERF1, an upstream component of JA and ET signaling, contributes to pathogen resistance . Transgee Tucker . Similarsicicola . In our AP24 and PR2 in the chloroplast enhanced resistance against filamentous pathogens  in Pi07586 transgenic lines (Pathogenesis-related (PR) proteins play a crucial role in plants\u2019 innate immune response to biotic and abiotic stress . For insathogens . The ant in vivo . The antnfestans . In our ic lines . NotablyMYC2 (Soltu.DM.06G022820.1) of the JA pathway was downregulated expression in Pi07586 transgenic lines  can be found below: YX: Writing \u2013 review & editing, Investigation, Writing \u2013 original draft. DZ: Investigation, Writing \u2013 original draft, Methodology. SC: Writing \u2013 original draft, Formal Analysis. LY: Formal Analysis, Writing \u2013 original draft, Resources. DeZ: Resources, Writing \u2013 original draft. HW: Funding acquisition, Supervision, Writing \u2013 review & editing."
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